Crystals of membrane proteins

ABSTRACT

A polypeptide in crystalline form comprises a G-protein coupled receptor (GPCR) with an IC3 loop substituted by an amino acid residue sequence of lysozyme.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/199,611 filed onSep. 2, 2011 which is a continuation of U.S. Ser. No. 12/739,134 filedon Sep. 23, 2010 which is a U.S. National Stage of PCT/US2008/080847,filed Oct. 22, 2008, and claims benefit of U.S. provisional application60/999,951, filed Oct. 22, 2007; U.S. provisional application61/000,325, filed Oct. 24, 2007; U.S. provisional application61/060,107, filed Jun. 9, 2008; and U.S. provisional application61/194,961, filed Oct. 1, 2008, each of which is incorporated herein byreference, in its entirety, for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. GM73197awarded by the National Institutes of Health; GM74691 and GM62411awarded by the Protein Structure Initiative; Y1-CO-1020 awarded by theNational Cancer Institute; and Y1-GM-1104 awarded by the NationalInstitute of General Medical Sciences. The government has certain rightsin this invention.

Coordinates and structure factors have been deposited in the ProteinData Bank with identification code 2RH1.

BACKGROUND OF THE INVENTION

Naturally occurring polypeptides or proteins often fold into complex,three-dimensional shapes that determine both chemical and physiologicalfunctionality. Thus a thorough understanding of proteins necessarilyinvolves a detailed representation of their spatial topography. Thefield of protein crystallography has flourished over the last 20 yearsresulting in a rapid increase in the knowledge bas of protein structureenabling great strides in other disciplines including biochemistry,pharmaceutical development and cell biology. However, the structuralbiology field has largely been restrained to working with protein thatis naturally soluble in aqueous media, or made soluble by incorporationinto surfactant micelles. The present invention provides methods andcompositions that allow for the study of membrane-embedded proteins(i.e., integral membrane proteins) in a more natural membrane bilayerenvironment. The present invention enable a more detailed analysis ofimportant classes of membrane-embedded polypeptides that play key rolesin a variety of cellular processes including energy and signaltransduction.

SUMMARY OF THE INVENTION

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this detaileddescription.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference for all purposes andto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

In one aspect the invention provides compositions, e.g., useful for thecrystallization of membrane protein. In some embodiments, thecomposition is suitable for lipidic cubic phase crystallization. In someembodiments the compositions contain 10-60% v/v of a polyethyleneglycol, 0.01-0.5 M of a salt, 1-20% v/v of an organic compound, and1-50% w/w of a lipid additive in a host lipid. In some embodiments ofthe compositions, the protein to be crystallized is present at aconcentration of 1 to 100 mg/mL. In some embodiments, the protein to becrystallized is present at a concentration of 50 mg/mL.

In some embodiments of the compositions of the inventions, thepolyethylene glycol is PEG or modified PEG at a molecular size of10-8000. In some embodiments, the PEG or modified PEG has an averagemolecular weight of 400-8000. In some embodiments, the PEG or modifiedPEG has an average molecular weight of 400-2000. In some embodiments,the PEG or modified PEG has an average molecular weight of 400-1000. Insome embodiments, the PEG or modified PEG has an average molecularweight of 400. Examples of modified PEG include but are not limited toPEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEGdistearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glycerylstearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenatedcastor oil, PEG castor oil, PEG corn oil, PEG caprate/caprylateglycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phytosterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEGsorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octylphenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester andmethoxypoly(ethylene glycol) (mPEG).

In some embodiments of the compositions, the salt is a sulfate salt. Insome embodiments, the sulfate salt is sodium sulfate. In someembodiments, the salt is present at a concentration of 0.1-0.5 M. Insome embodiments, the salt is present at a concentration of 0.1-0.2 M.In some embodiments, the compositions of the invention contain a buffer.In some embodiments, the buffer is present at a concentration of0.01-0.5 M. In some embodiments, the buffer is present at aconcentration of 0.1-0.2 M. In some embodiments, the buffer is presentat a concentration of 0.1 M. In some embodiments, the buffer is Bis-trispropane. In some embodiments, the buffer has a pH 6.5-7.0. In someembodiments of the compositions, an organic compound is present at aconcentration of 1-10% v/v. In some embodiments, the organic compound ispresent at a concentration of 5-7% v/v. In some embodiments, the organiccompound is 1,4-butanediol.

In some embodiments of the compositions, the lipid additive is presentat a concentration of 1-20% w/w in a host lipid. In some embodiments,the lipid additive is present at a concentration of 8-10% w/w in a hostlipid. Examples of lipid additives include but are not limited tocholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments,the lipid additive is cholesterol. Examples of host lipids include, butare not limited to monopalmitolein, monovaccenin and monoolein. In someembodiments, the host lipid is monoolein.

In some embodiments of this aspect, the composition of the inventioncomprises 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-trispropane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterolin monoolein as the host lipid.

In another aspect the invention includes compositions suitable forlipidic cubic phase crystallization. In some embodiments, thecompositions of the inventions contain a lipid additive. In someembodiments, the lipid additive is present at a concentration of 1-50%w/w in a host lipid. In some embodiments, the lipid additive is presentat a concentration of 1-20% w/w in a host lipid. In some embodiments,the lipid additive is present at a concentration of 8-10% w/w in a hostlipid. Examples of lipid additives include, but are not limited to,cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments,the lipid additive is cholesterol. Examples of host lipids include, butare not limited to monopalmitolein, monovaccenin and monoolein. In someembodiments the host lipid is monoolein.

In another aspect the invention includes methods for crystallization ofmembrane proteins. In some embodiments, the method for crystallizationof membrane proteins comprises adding a lipid additive to a lipidiccubic phase. Examples of lipid additives include, but are not limitedto, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In someembodiments, the lipid additive is cholesterol. In some embodiments, thelipid additive is present at a concentration of 1-50% w/w in a hostlipid. In some embodiments, the lipid additive is present at aconcentration of 1-20% w/w in a host lipid. In some embodiments, thelipid additive is present at a concentration of 8-10% w/w in a hostlipid. Examples of host lipids include, but are not limited tomonopalmitolein, monovaccenin and monoolein. In some embodiments thehost lipid is monoolein.

In another aspect, the invention provides for methods of crystallizationof a protein. In some embodiments of this aspect, the method comprises,providing said protein in a lipidic cubic phase composition, filling aplate comprising a material that does not interfere with imaging (suchas, e.g., a transparent glass or plastic) with said composition, placingsaid plate containing said composition under conditions suitable forcrystallization of said protein and detecting the presence of a crystalof said protein in said plate. In some embodiments, the method furthercomprises covering said plate with a second plate comprising a materialthat does not interfere with imaging (such as, e.g., a transparent glassor plastic).

In some embodiments of the methods, the protein is a non-coloredprotein. In some embodiments, the protein is a G protein-coupledreceptor (GPCR). In some embodiments, the protein comprises a β₂AR, aCXCR4, or a human adenosine A_(2A) receptor. In some embodiments theprotein comprises a stabilizing point mutation or a T4 lysozyme fusionor both.

In some embodiments of the methods, the lipidic cubic phase compositioncomprises a lipid additive. Examples of lipid additives include, but arenot limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In someembodiments, the lipid additive is cholesterol. In some embodiments, thelipid additive is present at a concentration of 1-50% w/w in a hostlipid. In some embodiments, the lipid additive is present at aconcentration of 1-20% w/w in a host lipid. In some embodiments, thelipid additive is present at a concentration of 8-10% w/w in a hostlipid. Examples of host lipids include, but are not limited tomonopalmitolein, monovaccenin and monoolein. In some embodiments thehost lipid is monoolein.

In some embodiments of the methods, the first plate and second plate aremade of glass. In some embodiments, the plate allows for the control ofcrystallization conditions, such as the humidity and temperature of saidlipidic cubic phase composition.

In some embodiments of the methods, the crystals are harvested directlyfrom the plate. In some embodiments of the methods, the crystals areharvested between the cubic and the sponge phase of the lipidic cubicphase composition. In some embodiments of the methods, the crystals areharvested directly from said lipidic cubic phase composition and placingsaid crystals in liquid nitrogen.

In another aspect the invention provides methods for screening a crystalpresent in a liquid cubic phase composition. In some embodiments, themethod comprises exposing the composition to a first beam anddetermining a change of the first beam, exposing the composition to asecond beam and determining a change of the second beam, and identifyingan area where the crystal is present in said composition. Examples ofchanges in the beams, include but are not limited to, change indirection and/or intensity of the beams. In some embodiments thecrystals are non-colored.

In some embodiments of the methods, the first beam and second beam areattenuated. In some embodiments the beams are attenuated 10 times. Insome embodiments, the first beam is a stilted 100×25 μm beam. In someembodiments, the methods comprise exposing said lipidic cubic phasecomposition to a third beam. In some embodiments, the methods compriseexposing said lipidic cubic phase composition to up to ten extra beams.In some embodiments, the exposure of composition to the beams is 2seconds. In some embodiments, the beams are beams of visible light.

In another aspect the invention includes a crystal of a membraneprotein. In some embodiments, the invention includes a crystal of a Gprotein-coupled receptor (i.e., a “GPCR”) non-covalently bound to aligand. In some embodiments, the extracellular domain of saidcrystalline GPCR is resolvable by X-Ray crystallography. In someembodiments, the ligand is a diffusible ligand.

In some embodiments of this aspect, the volume of the crystal is greaterthan 15×5×1 μm. In some embodiments, the volume of said crystal isgreater than 30×5×5 μm. In some embodiments of this aspect, the volumeof crystal is greater than 40×20×5 μm. In some embodiments, volume ofthe crystal is estimated assuming that each of the stated dimensions areorthogonal so that the volume estimate is the product of the dimensions.In some embodiments, the crystal is suitable for X-ray crystallography.In some embodiments, X-ray crystallographic analysis can be carried outto determine the structure of a protein comprising said crystal.

In some embodiments of this aspect, the crystal is crystallized usingliquid cubic phase crystallization. In some embodiments, the crystal isobtainable by harvesting the crystal from a glass sandwich plate. Insome embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å.In some embodiments, the crystal diffract to a resolution of 2.0 to 5.0Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å.In some embodiments, the structure of said crystal is solved and refinedat resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In someembodiments, the structure of said crystal is solved and refined atresolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments,the structure of said crystal is solved and refined at resolution ofless than about 2.4 Å.

In some embodiments, the G protein-coupled receptor is a β₂AR protein, aCXCR4 protein, or a human adenosine A_(2A) receptor protein.

In another aspect the invention provides for a crystal of β₂AR. In someembodiments, the structure of an extracellular domain of said crystal iscapable of being resolved by X-ray crystallography. In some embodiments,the crystal comprises 442 amino acids, a palmitic acid covalently boundto Cys341 and an acetamide molecule bound to Cys265^(6.27), a diffusibleligand, up to 10 molecules a lipid additive, up to five salt ions and upto 10 butanediol molecules. In some embodiments, the lipid additive ischolesterol. In some embodiments, the crystal comprises threecholesterol molecules. In some embodiments, the salt ion is a sulfateion. In some embodiments, the crystal comprises two sulfate ions. Insome embodiments, the diffusible ligand is carazol. In some embodimentsthe crystal comprises two butanediol molecules. In some embodiments ofthis aspect, the volume of the crystal is greater than 15×5×1 μm. Insome embodiments, the volume of said crystal is greater than 30×5×5 μm.In some embodiments of this aspect, the volume of the crystal is greaterthan 40×20×5 μm. In some embodiments the volume of the crystal isestimated by assuming that each dimension is orthogonal to the otherdimensions so that the volume is the product of the three lengths. Insome embodiments, the crystal is suitable for X-ray crystallography. Insome embodiments, the structure of a β₂AR protein can be determined fromsaid crystal using X-ray crystallographic analysis. In some embodiments,the crystal is crystallized using liquid cubic phase crystallization. Insome embodiments, the crystal is obtainable by harvesting the crystalfrom a glass sandwich plate. In some embodiments of this aspect, thecrystal is crystallized using liquid cubic phase crystallization. Insome embodiments, the crystal is obtainable by harvesting the crystalfrom a glass sandwich plate. In some embodiments, the crystal diffractto a resolution of 1.0 to 10.0 Å. In some embodiments, the crystalsdiffract to a resolution of 2.0 to 5.0 Å. In some embodiments, thecrystal diffract to a resolution of 2.2 Å. In some embodiments, thestructure of said crystal is solved and refined at resolution of lessthan about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure ofsaid crystal is solved and refined at resolution of less than about 2.8,2.6 or 2.4 Å. In some embodiments, the structure of said crystal issolved and refined at resolution of less than about 2.4 Å.

In another embodiment, the invention provides a composition for lipidiccubic phase crystallization of a membrane protein, comprising apolyethylene glycol or modified polyethylene glycol; 0.01-1M of a salt;a host lipid; a lipid additive, wherein said lipid additive is presentat 10-60% v/v ratio relative to the host lipid; a buffer; and 1 to 100mg/ml of a membrane protein. In a related embodiment, the polyethyleneglycol is PEG or modified PEG, wherein said PEG or modified PEG has anaverage molecular weight of 200-20,000, 400-8000, or 400-2000. In yetanother related embodiment, the PEG or modified PEG in the compositionhas an average molecular weight of 400. In another related embodiment,the salt is selected from the group consisting of a sulfate salt, acitrate salt, a malonate salt, a tartrate salt, an acetate salt, and aformate salt. In certain embodiments of the composition, the salt ispresent at a concentration of 0.1-0.2 M. In another related embodiment,the buffer is present at a concentration of 0.05-0.5 M in thecomposition. In certain embodiments, the buffer is Bis-tris propane orsodium citrate. In other related embodiments of the composition, thebuffer has a pH between 4.5-8.0.

In still other related embodiments of the composition for lipidic cubicphase crystallization of a membrane protein, the composition furthercomprising an alcohol present at a concentration of 1-10% v/v or 5-7%v/v. In certain embodiments, the alcohol is a diol or triol. In otherembodiments, the alcohol is 1,4-butanediol or 2,6-hexanediol.

In still other related embodiments of the composition for lipidic cubicphase crystallization of a membrane protein, the lipid additive ispresent at a concentration of 1-20% w/w in a host lipid or 8-10% w/w ina host lipid. In yet another related embodiment of the composition, thelipid additive is selected from the group consisting of 2-monoolein,phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid,dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoylphosphatidylcholine (“DOPE”), Asolectin, and a sterol. In still otherembodiments, the lipid additive is a sterol. In related embodiments, thelipid additive is cholesterol.

In still another related embodiment of the composition for lipidic cubicphase crystallization of a membrane protein, the host lipid is selectedfrom the group consisting of monopalmitolein, monovaccenin andmonoolein. In a related embodiment, the host lipid is monoolein. Instill another related embodiment, the membrane protein to becrystallized in said composition is present at a concentration of 1 to100 mg/mL. In yet another embodiment, the membrane protein to becrystallized in said composition is present at a concentration of 40-60mg/mL.

In still another related embodiment of the composition for lipidic cubicphase crystallization of a membrane protein membrane protein is aG-protein coupled receptor, such as a β₂AR protein, a human adenosineA2A receptor protein, a CXCR4-T4L protein, or a (β₂AR-T4L protein. Inrelated embodiments, the G-protein coupled receptor is a proteincomprising or consisting of a β₂AR^((E122W)), a β₂AR^((E122W))-T4L, ahuman adenosine A2A receptor-T4L, a CXCR4-T4L or β₂AR-T4L. In stillanother related embodiment of the composition for lipidic cubic phasecrystallization of a membrane protein, the composition comprises aligand selected from the group consisting of carazolol, timolol,alprenolol, and clenbutorol.

In another embodiment, the invention provides a method of generatingcrystals of a membrane protein comprising: mixing a lipid additive witha host lipid to form a lipid mixture, wherein said lipid additive isselected from the group consisting of a sterol, DOPE, DOPE-Me, DOPC, andAsolectin, and wherein said lipid additive is 5 to 20% w/w in said hostlipid; and combining said lipid mixture with a membrane protein solutionunder conditions suitable for the formation of a lipidic cubic phasecomposition. In a related embodiment of the method, said protein is anon-colored protein. In a related embodiment, the amount of said lipidadditive is 8 to 10% w/w in said lipid. In another related embodiment,the invention further comprises: filling a plate with said lipidic cubicphase composition, wherein said plate is compatible with imaging;placing said plate containing said lipic cubic phase composition underconditions suitable for crystallization of said protein; and detectingthe presence of a crystal of said protein in said plate. In anotherembodiment, the method further comprises covering said plate with asecond plate.

In a related embodiment of the method of generating crystals of amembrane protein, the protein is a GPCR. In yet another relatedembodiments, the protein comprises a β₂AR. In yet another related)embodiment, the. β₂AR protein is selected from the group consisting ofβ₂AR^((E122W)), β₂AR^((E122W))-T4L, and β₂AR-T4L. In yet another relatedembodiment, the GPCR is a human adenosine A2A receptor or a CXCR4receptor where the proteins may comprise, in still other relatedembodiments, a T4 lysozome.

In yet another related embodiment of method of generating crystals of amembrane protein, the lipid additive is present at a concentration of1-20% w/w or 8-10% w/w in a host lipid. In yet another relatedembodiment of the method, the second plate comprises a glass. In yetanother related embodiment, the method further comprises harvestingcrystals directly from said plate. Another related embodiment of themethod comprises harvesting crystals from between the cubic and thesponge phase of said lipidic cubic phase composition. In another relatedembodiment, the method comprises harvesting crystals directly from saidlipidic cubic phase composition and placing said crystals in liquidnitrogen. In yet another related embodiment, the method comprising astep of soaking into said crystal a diffusible ligand or candidateligand.

The invention also provides a method of screening a crystal of a GPCRpresent in a liquid cubic phase composition comprising: preparing aliquid cubic phase composition comprising a GPCR protein, a host lipid,and a lipid additive; exposing said composition to a first X-ray beamand determining a change in direction or intensity of said first X-raybeam; exposing said composition to a second beam and determining achange in direction or intensity of said second X-ray beam; identifyingan area where said GPCR crystal is present in said composition; andexposing said identified area to at least a third X-ray beam. In arelated embodiment, the first beam is a slitted 100×25 μm beam. Inanother related embodiment, the crystal is colorless. In yet anotherembodiment, the GPCR crystal is β₂AR^((E122W))-T4L, β₂AR^((E122W)),β₂AR, or β₂AR-T4L protein. In related embodiments, the crystal is humanadenosine A2A receptor or a CXCR4 receptor where the proteins maycomprise, in still other related embodiments, a T4 lysozome.

In another embodiment, the invention provides a crystal of a human β₂ARprotein wherein the extracellular loop ECL2 of said β₂AR is sufficientlyordered to produce interpretable electron density in acrystallographically-derived electron density map. In yet anotherrelated embodiment, each β₂AR molecule in said crystal comprises threenon-covalently bound cholesterol molecules and at least one salt ion. Inyet another related embodiment, the at least one salt ion is a sulfateion. In yet another related embodiment, each β₂AR molecule in saidcrystal said crystal comprises two sulfate ions. In yet another relatedembodiment, each β₂AR molecule in said crystal said crystal furthercomprises carazol. In yet another related embodiment of the crystal,each β₂AR molecule in said crystal comprises between 1 and 10 butanediolmolecules. In yet another related embodiment of the crystal, the volumeof said crystal exceeds 15×5×1 μm, 30×5×5 μm or 40×20×5 μm. In yetanother related embodiment, the specific surface area of the crystal is0.0001-5 m²/g. In yet another related embodiment, the crystal iscrystallized using liquid cubic phase crystallization. In yet anotherrelated embodiment, the crystal is obtainable by harvesting the crystalfrom a glass sandwich plate. In yet another related embodiment, thecrystal diffracts to a resolution of 2.0 to 10.0 Å, 2.0 to 5.0 Å, or 2.2to 2.4 Å. In yet another related embodiment, the structure of saidcrystal is solved and refined at a resolution higher than about 3.2,higher than about 2.8, or higher than about 2.4 Å.

In yet another embodiment, the invention provides a crystalline form ofa human β₂AR protein having an atomic arrangement of coordinatescomprising the β₂AR coordinates set forth in Appendix I (SEQ ID NOS 4-5,1 and 6-9, respectively in order of appearance).

In another embodiment, the invention provides a crystalline form of ahuman β₂AR protein, where said form has unit cell dimensions of a=106.3Angstroms, b=169.2 Angstroms, and c=40.2 Angstroms. In arelatedembodiment, said β₂AR protein is β₂AR-T4L. In another relatedembodiment, the β₂AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of ahuman β₂AR protein, wherein said space group of said crystalline form isC2. In a related embodiment, said β₂AR protein is β₂AR-T4L. In anotherrelated embodiment, the β₂AR-T4L crystal further comprises a carazololligand.

In another embodiment, the invention provides a crystalline form of ahuman β₂AR protein, wherein said crystalline form diffracts X-rays to aresolution of 2.4 Angstroms. In a related embodiment, said β₂AR proteincomprises a point mutation that stabilizes said β₂AR protein. In anotherrelated embodiment, said β₂AR protein is β₂AR-T4L. In a relatedembodiment, the β₂AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of ahuman β₂AR protein wherein each β₂AR molecule in said crystal comprises442 amino acids, a palmitic acid covalently bound to Cys341, anacetamide molecule bound to Cys265^(6.27), a diffusible ligand, one toten molecules of a lipid additive, one to five salt ions and one to tenbutanediol molecules.

In another embodiment, the invention provides a method of identifying acompound that binds to a ligand binding site of a human β₂AR protein,comprising comparing a set of three-dimensional structures representinga set of candidate compounds with a three-dimensional molecular model ofsaid ligand binding site, comprising: receiving a three-dimensionalmodel of a ligand binding site on said human β₂AR protein, wherein saidthree-dimensional model of said ligand binding site comprises atomicco-ordinates for a plurality of ligand-binding residues, wherein saidatomic co-ordinates are taken from Appendix I (SEQ ID NOS 4-5, 1 and6-9, respectively in order of appearance); determining, for each of theset of compound three-dimensional models, a plurality of distance valuesindicating distances between said atomic co-ordinates of said candidatecompound of the set of candidate compounds and said atomic coordinatesof said ligand-binding residues comprising said ligand binding site;determining, for each of the set of compound three-dimensional models, abinding strength value based on the plurality of distance valuesdetermined for the compound three-dimensional model, wherein the bindingstrength value indicates the stability of a complex formed by said humanβ₂AR protein and a compound represented by the compoundthree-dimensional model; and storing a set of results indicating whethereach candidate compound binds to the three-dimensional model based onthe binding strength values. In a related embodiment, saidligand-binding residues comprise a plurality of residues selected fromthe group consisting of Y199, A200, S204, T118, V117, W286, Y316, F290,F289, N293, W109, F193, and Y308. In another related embodiment, saidligand-binding residues comprise a plurality of residues selected fromthe group consisting of W109, V117, T118, F193, Y199, A200, W286, F289,F290, Y316. In another related embodiment of the method of identifying acompound that binds to a ligand binding site of a human β₂AR protein,said binding strength value is based on one or more of a hydrogenbonding strength, a hydrophobic interaction strength, or a Coulombicinteraction binding strength. In another related embodiment, one or moreof said receiving, determining, or storing steps is carried out using acommercially-available software program. In yet another relatedembodiment, the commercially-available software program is selected fromthe group consisting of DOCK, QUANTA, Sybyl, CHARMM, AMBER, GRID, MCSS,AUTODOCK, CERIUS II, Flexx, CAVEAT, MACCS-3D, HOOK, LUDI, LEGEND,LeapFrog, Gaussian 92, QUANTA/CHARMM, Insight II/Discover, and ICM. Inyet another related embodiment, the method further comprising the stepof contacting a human β₂AR protein with a molecule comprising anidentified candidate compound. In yet another related embodiment, themolecule further comprises a moiety capable of competitively displacinga ligand from said human β₂AR protein, wherein said ligand binds to saidligand binding site of said human β₂AR protein. In yet another relatedembodiment, the method further comprising characterizing a bindinginteraction between said human β₂AR protein and said molecule comprisingsaid identified candidate compound, and storing a result of saidcharacterizing. In yet another related embodiment, said characterizationcomprises determining an activation of a function of said human β₂ARprotein, an inhibition of a function of said human β₂AR protein, anincrease in expression of said human β₂AR protein, a decrease inexpression of said human β₂AR protein, a displacement of a ligand boundto said ligand binding site, or a stability measure for said human β₂ARprotein.

In another embodiment, the invention provides a method for selecting alibrary of potential modulators of β₂AR to be screened, comprisingcalculating a structure of a first potential modulator using at least aportion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1and 6-9, respectively in order of appearance), correlating saidstructure of said first potential modulator with a library of modulatorsidentified as comprising said structure said first potential modulator,and storing or transmitting information about the identified library.

In yet another embodiment, the invention provides a method of solvingthe structure of a crystalline form of a protein, comprising: using atleast a portion of the structure co-ordinates of Appendix I (SEQ ID NOS4-5, 1 and 6-9, respectively in order of appearance) to solve thestructure of the crystalline form of a test protein, wherein said testprotein has significant amino acid sequence homology to any functionaldomain of β₂AR; and transmitting or storing data descriptive of thestructure of said test protein.

In another embodiment, the invention provides a method of identifyingfrom a set of candidate compound three-dimensional models a compoundthat binds to a ligand binding site of a GPCR or β₂AR proteincomprising: receiving a three-dimensional model of a ligand binding siteon said GPCR or β₂AR protein, wherein said three-dimensional model ofsaid ligand binding site comprises atomic co-ordinates for a pluralityof ligand binding residues; determining, for each candidate compound ofthe set of candidate compound three-dimensional models, a plurality ofdistance and angle values indicating distances and angles between atomicco-ordinates of said candidate compound of the set of candidate compoundthree-dimensional models and said ligand binding site comprising atomiccoordinates of said ligand-binding residues; determining, for each ofthe set of candidate compound three-dimensional models, a bindingstrength value based on the plurality of distance and angle valuesdetermined for the candidate compound three-dimensional model, whereinthe binding strength value indicates the stability of a complex formedby said human GPCR or β₂AR protein and a compound represented by thecompound three-dimensional model; storing a set of results indicatingwhether each candidate compound binds to the three-dimensional modelbased on the binding strength values; searching a database of smallorganic molecules for compounds exhibiting shape, chemistry, orelectrostatic similarity with the candidate compounds indicated to bindto the three-dimensional model based on the binding strength values; andidentifying the set of small organic molecules exhibiting shape,chemistry, or electrostatic similarity with the candidate compoundsindicated to bind to the three-dimensional model based on bindingstrength values as likely to also bind to the GPCR or β₂AR.the databaseof small organic molecules is the available chemicals database. In arelated embodiment, the shape, chemistry or electrostatic similarity isdetermined using a program selected from the group consisting of BROOD(openeye), EON (openeye), ROCS (openeye), ISIS Base, and SciFinder.

In another embodiment, the invention provides a method of identifying aligand that binds to a membrane protein comprising: preparing a lipidmeso phase, wherein said lipid meso phase composition comprises (1) ahost lipid; (2) said membrane protein; (3) a lipid additive selectedfrom the group consisting of consisting of a sterol, cholesterol, DOPE,DOPE-Me, DOPC, and Asolectin, wherein said lipid additive is 1 to 50%w/w in a lipid host; subjecting said lipid meso phase to humidity andtemperature conditions to grow crystals of said membrane protein;contacting said membrane protein crystals with diffusible ligands or amixture of diffusible ligands; determining the three-dimensionalstructure of said diffusible ligand contacted membrane protein crystalsby X-ray crystallography to obtain an electron density map; andidentifying bound ligands by inspection of the electron density map. Ina related embodiment, the ligands are substantially insoluble in water.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of the necessary fee.

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1. Crystals of β₂AR-T4L obtained in 30-35% v/v PEG400, 0.1-0.2 M Nasulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediolusing 8-10% w/w cholesterol in monoolein as the host lipid. FIG. 1 ashows β₂AR-T4L crystals in the crystallization mixture drop (upper left)and in the loop. FIG. 1 b shows crystals in the “sponge” phase.

FIG. 2. Before (top) and after (bottom) images of a lipidic cubic phasecrystal harvested directly from a well in a previously sealed glasssandwich plate, according to the method described herein (see, e.g.,Example 1).

FIG. 3. Diffraction pattern (2.8 {acute over (Å)} resolution) fromβ₂AR-T4L crystals grown in lipid cubic phase. The crystal size wasapproximately 25×5×5 {acute over (Å)}; space group C2 (a=106.8 {acuteover (Å)}, b=169.5 {acute over (Å)}, c=40.5 {acute over (Å)}; β=105.3°,α=γ=90°. Beam diameter was 10 μm, exposure 10 s, oscillation: 1°.

FIG. 4. Gallery of crystals of various GPCRs obtained usingLCP/cholesterol mixtures and in combination with a variety of ligands.The top panel corresponds to non-optimized initial hits, whereas thebottom panel shows diffraction quality for optimized crystals. From leftto right, β₂AR-T4L (bound to carazolol), diffracted to 2.4 Å resolution;β₂AR(E122W)T4L (bound to carazolol), diffracted to 3.5 Å resolution;β₂AR(E122W)T4L (bound to alprenolol), diffracted to 3.5 Å resolution;β₂AR(E122W)T4L (bound to timolol), diffracted to 2.8 Å resolution;β₂AR(E122W) (bound to carazolol); β₂AR(E122W)T4L (bound to clenbuterol),diffracted to 6 Å, anisotropic; human A_(2A) adenosine receptor-T4L(bound to ZM241385), diffracted to 2.6 Å resolution.

FIG. 5. A. β₂AR-T4L Crystals grown from bicelle conditions. B.Diffraction image from bicelle grown microcrystals of β₂AR-T4L recordedusing 10 μm minibeam on 231D-B beamline at APS. Black circle is drawn atresolution 3.5 Å.

FIG. 6. A. Microcrystals of β₂AR-T4L grown in lipidic mesophase. B.Diffraction image from lipidic cubic phase grown microcrystals ofβ₂AR-T4L recorded using a 10 μm minibeam on 231D-B beamline at APS. Thewhite circle is drawn at resolution 2.2 Å.

FIG. 7. Detailed representation of the carazolol binding site inβ₂AR-T4L. FIGS. 7A, B, and C are representations of the electron densityof the ligand binding site at three different orientations. Residues arelabeled by their Ballesteros-Weinstein numbers as superscripts. Electrondensity is contoured at 16 from a 2 F_(o)-F_(c) difference map. Both Band C are generated by rotating the field of view 90° about the y-axisclockwise and counterclockwise respectively.

FIG. 8. Electron density of: A. Cholesterol molecules shown with aF_(o)-F_(e) electron density contoured at 2σ omitting the lipid fromphase calculation, palmitic acid is also shown. B. Helix-kinked regionwith 2F_(o)-F_(c) electron density contoured at 1.5σ.

FIG. 9. Overall fold of the β₂AR-T4L fusion with its predictedorientation in the plasma membrane and key intramolecular interactions.A. Stereoview of the overall fold of β₂AR-T4L. The receptor and T4L arecolored gray and green, respectively. Carazolol is colored blue and thelipid molecules bound to the receptor are colored yellow. B. Thereceptor is aligned to a rhodopsin model that was positioned in a lipidmembrane (boundaries indicated by horizontal black lines) as found inthe orientations of proteins in membranes (OPM) database (M. A. Lomizeet al., Bioinformatics 22, 623 (2006)). T4L is fused internally into thethird intracellular loop of β₂AR and maintains minimal intramolecularpacking interactions by tilting away from the receptor. C. Specificintramolecular interactions between β₂AR and T4L are represented.

FIG. 10. Crystal packing interactions in the lipidic mesophasecrystallized β₂AR-T4L. A. There are four main contact areas, two ofwhich are mediated by T4L in the plane of the membrane with itselfthrough a two-fold symmetry axis and translation. The third interactionis normal to the membrane plane between T4L and lumen exposed loops ofβ₂AR. The fourth interaction is generated by the two-fold symmetry axis,packing one receptor to receptor in the plane of the membrane. B. Thereceptor crystal packing interface is composed mainly of lipids with twocholesterol molecules and two palmitic acid molecules forming themajority of the interactions. A network of ionic charge interactionsexists on the cytoplasmic end of the interface forming the onlyinter-receptor protein contacts. C. Comparison between β₂AR-T4L andrhodopsin (PDB ID Code 2135) parallel receptor association interface.Helices I (blue) and VIII (magenta) are highlighted in both structures.Only one monomer is shown for each receptor representation along withhelices I′ and VIII′ only from the opposing symmetry related molecule.The rhodopsin interface is twisted significantly relative to β2AR-T4Lresulting in a significant offset from the parallel orientation requiredfor a physiological dimer interface. β₂AR-T4L associated monomers are ina highly parallel orientation.

FIG. 11. Surface representation of β₂AR colored by calculated chargefrom red (−10 k_(b)T/e_(c)) to blue (+10 k_(b)T/e_(c)) using adielectric constant of 70. A. Three main areas of interest areindicated. The binding site cleft is negatively charged as is a groovebetween helices III, IV and V. The third region is an overall positivecharge in the region of the ionic lock and DRY motif on the cytoplasmicface. The overall result is a highly polarized molecule that may utilizeits negative charge to facilitate binding of catecholamine ligands. Thepresence of a negative charge in the groove between helices III, IV andV is unexpected as it is in the middle of the lipid membrane. Thischarge may be partially derived from the presence of an unpairedglutamate at position 122^(3.41). The effective charge in this region islikely greater than shown here due to its location in the low dielectricenvironment of the lipid membrane. B. View rotated 90° from A. Showingboth the negatively charged binding site cleft (top) and positivelycharged cytoplasmic face (bottom). Poisson-Boltzmann electrostatics werecalculated using the program APBS (Baker et al., Proc Natl Acad Sci U SA, 98, 10037 (2001)) as implemented in Pymol (The PyMOL MolecularGraphics System (2002) on World Wide Web http://www.pymol.org). Pymolwas used exclusively in the preparation of all figures.

FIG. 12. Comparison of the extracellular sides of β₂AR-T4L andrhodopsin. A. The N-terminus is missing from the experimental density inthe β₂AR-T4L structure and is not shown. ECL2 is shown in green andcontains a short α-helix and two disulfide bonds (yellow). The intraloopdisulfide bond constrains the tip of ECL2 which interacts with ECL1. Thesecond disulfide bond links ECL2 with helix III. There is oneinteraction between ECL2 and carazolol (blue) through Phe193^(5.32). Theentire loop is held out of the ligand binding site by a combination ofthe rigid helical segment and the two disulfide bonds. B. In contrast,ECL2 (green) in rhodopsin assumes a lower position in the structure thatoccludes direct access to the retinal-binding site and forms a smallβ-sheet in combination with the N-terminal region (magenta) directlyabove the bound retinal (pink).

FIG. 13. Ligand binding characterization and comparison to rhodopsin. A.A view looking down on the plane of the membrane from the extracellularsurface showing a detailed representation of the carazolol binding sitein β₂AR-T4L. Carazolol is shown as sticks with carbon atoms coloredyellow. β₂AR-T4L residues contributing to carazolol binding are shown ingreen and labeled. Electron density is contoured at 5σ from anF_(o)-F_(c) omit map calculated without the contribution of carazolol.B. Binding orientation comparison between 11-cis-retinal in rhodopsinand carazolol in β₂AR-T4L. Van der Waals' surfaces for carazolol andretinal are represented as dots to accentuate the close packinginteractions. Retinal in the all-cis conformation (pink), binds deep inthe active site of rhodopsin as compared to carazolol (blue), packingits β-ionone ring between Tyr268^(6.51) and Phe212^(8.47) (cyan),blocking movement of Trp265^(6.48) (magenta) into the space. Theβ-ionone ring of trans-retinal in activated rhodopsin would not blockTrp265^(6.48) from rotating into the space allowing a rotameric shiftinto its proposed active form. C. There are four residues involved inthe toggle switch mechanism of β₂AR-T4L as shown. Phe290^(6.52)(magenta) is sandwiched between Phe208^(5.47) (tan) and Phe289^(6.51)(tan) forming a ring-face aromatic interaction. Like rhodopsin, anactivation step is thought to occur by a rotameric change ofTrp286^(6.48) (magenta) which would displace Phe290^(6.52). Carazolol isshown to interact extensively with the sandwich motif as shown: however,few interactions are seen with Trp286^(6.48). The 6.52 position inβ₂AR-T4L is occupied by Phe290^(6.82) as opposed to Ala269^(6.82) inrhodopsin where the β-ionone ring replaces an aromatic protein sidechain in forming the sandwich interactions. The aromatic character ofthe sandwich is otherwise maintained by Phe289^(6.81) and Phe208^(8.47)in β₂AR-T4L.

FIG. 14. Comparison of β₂AR-T4L helical orientations with rhodopsin (PDBID Code 1U19). A. β₂AR-T4L is rendered as a ribbon trace colored with ablue to red spectrum corresponding to observed distances between Cαpositions in the two structures (RMSD 2.7 Å between all residues in thetransmembrane region). Helix II shows very little movement, whereas theentire lengths of helices III, IV, V shift significantly. Helix VIII andloops were not included in the comparison and are colored in tan. B.Movements of helices I and V of rhodopsin (grey) are shown relative toβ₂AR-T4L. C. Movements of helices III, IV and VI. D. Ligand binding siterepresentation. Carazolol is shown with yellow carbons. Entire helicesare assigned a single designation based on their divergence from therhodopsin position in the area of the ligand binding site as shown.Helix I is highly divergent, Helices II and VI are similar to rhodopsin.Helices IV and VII are moderately constant. Helices III and V aremoderately divergent.

FIG. 15. Affinity curves for adrenergic ligands binding to β₂AR-T4L andwildtype β₂AR. Saturation curves for the antagonist [³H]DHA is shown atleft, next to competition binding curves for the natural ligand(−)-Epinephrine and the high-affinity synthetic agonist Formoterol.Binding experiments on membranes isolated from Sf9 insect cellsexpressing the receptors were performed as described above.

FIG. 16. Comparison of the proteolytic stability between the wild-typeβ₂AR and β₂AR-T4L in a limited trypsin proteolysis assay. TPCK-trypsinwas added to carazolol-bound, purified, dodecylmaltoside-solubilizedreceptor at a 1:1000 ratio (wt:wt), and samples were analyzed bySDS-PAGE. Intact β₂AR-T4L (56.7 kD) and FLAG-tagged wild-type β₂AR (47.4kD) migrate similarly as ˜55 kD bands. Markers are Biorad low-rangeSDS-PAGE protein standards.

FIG. 17. Stability comparison of unliganded β₂AR365 and β₂AR-T4L. Fordodecylmaltoside-solubilized receptor preparations, maintenance of theability to specifically bind [³H]DHA after incubation at 37° C. is takenas a measure of stability.

FIG. 18. Superimposed Cα traces of the receptor component of β₂AR-T4L(in blue) and β2AR365 (in yellow). Common modeled transmembrane helixregions 41-58, 67-87, 108-137, 147-164, 204-230, 267-291, 312-326,332-339 were used in the superposition by the program Lsqkab (The CCP4Suite, Acta Crystallogr D Biol Crystallogr 50, 760 (1994)) (RMSD=0.8 Å).

FIG. 19. Carazolol dissociation from β₂AR365.Dodecylmaltoside-solubilized carazolol-bound receptor (at 50 μM) wasdialyzed in a large volume of buffer containing 300 micromolaralprenonol as a competing ligand, and aliquots were removed from thedialysis cassette at different time points. Remaining bound carazololwas measured (in a relative sense) by collecting fluorescence emissionwith excitation at 330 nm and emission from 335 to 400 nm. For eachcarazolol fluorescence measurement, data was normalized for the proteinconcentration in the dialysis cassette (measured with the Bio-RadProtein DC kit). The Y-axis represents carazolol fluorescence emissionIntensity (in cps) at 341 nm. The exponential decay of carazololconcentration in the receptor dialysis cassette was fit using GraphpadPrism software, giving a half-life of 30.4 hrs.

FIG. 20. Comparison of β₁ and β₂AR sequences. After aligning the β₁ andβ₂AR sequences, positions that have different amino acids between thetwo receptors were mapped onto the high-resolution structure of β₂ART4L(shown as red sticks). The carazolol ligand is shown as green sticks(with nitrogens in blue and oxygens in red). Highlighted residuesAla85^(2.56), Ala92^(2.63) and Tyr308^(7.35) are homologous to aminoacids Leu110^(2.56), Thr117^(2.63) and Phe359^(7.35) of the β₁AR, whichwere shown to be primarily responsible for its selectivity over β₂AR forthe compound RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51(2002)). In the β₂AR-T4L structure, only Tyr308^(7.35) faces the ligand,while Ala85^(2.56) lies at the interface between helices II and III. Ofall the divergent amino acids, only Tyr308^(7.35) is found within 4 Å ofany atom of carazolol.

FIG. 21. Design and optimization of the β₂AR-T4L fusion protein A. Thesequence of the region of the β₂AR targeted for insertion of acrystallizable domain is shown (SEQ ID NO:2), and the positions of thejunctions between the receptor and T4L (in red) for various constructsare indicated. The sequences that were initially replaced or removed arefaded. Red lines are shown after every tenth residue. Peptide ‘LNKYADWT’disclosed as SEQ ID NO: 3 B. Immunofluorescence images of HEK293 cellsexpressing selected fusion constructs. Panels on the left shows M1anti-FLAG signal corresponding to antibody bound to the N-terminus ofthe receptor. Panels on the right show the same signal merged with blueemission from DAPI (nuclear staining for all cells). Plasma membranestaining is observed in the positive control, D3 and D1, while C3 and D5are retained in the endoplasmic reticulum.

FIG. 22. Functional characterization of β₂AR-T4L. A. Affinitycompetition curves for adrenergic ligands binding to β₂AR-T4L andwild-type β₂AR. Binding experiments on membranes isolated from Sf9insect cells expressing the receptors were performed as described in themethods section of Example 4. B. β₂AR-T4L is still able to undergoligand-induced conformational changes. Bimane fluorescence spectra(excitation at 350 nm) of detergent-solubilized β₂AR-T4L and wild-typeβ₂AR truncated at 365, labeled under conditions that selectively modifyCys265^(6.27) (see methods section of Example 4), were measured afterincubating unliganded receptor with compounds for 15 min at roomtemperature. The cartoon illustrates that the observed changes influorescence can be interpreted as a movement of the bimane probe from amore buried, hydrophobic environment to a more polar, solvent-exposedposition.

FIG. 23. A. Side-by-side comparison of the crystal structures of theβ₂AR-T4L fusion protein and the complex between β₂AR365 and a Fabfragment. The receptor component of the fusion protein is shown as ablue cartoon (with modeled carazolol as red spheres), while the receptorbound to Fab5 is in yellow. B. Differences in the environmentsurrounding Phe264^(6.26) (shown as spheres) for the two proteins. C.The analogous interactions to the “ionic lock” between the E(D)RY motifand Glu247^(6.30) seen in rhodopsin (right panel, darkened) are brokenin both structures of the β₂AR (left panel). Pymol (W. L. DeLano, ThePyMOL Molecular Graphics System (2002) on the World Wide Webhttp://www.pymol.org) was used for the preparation of all figures.

FIG. 24. Schematic representation of the interactions between β₂AR-T4Land carazolol at the ligand binding pocket. Residues shown have at leastone atom within 4 Å of the ligand in the 2.4 Å resolution crystalstructure.

FIG. 25. The ligand binding pocket of β₂AR-T4L with carazolol bound. A.Residues within 4 Å of the ligand are shown as sticks, with theexception of A200, N293, F289, and Y308. Residues that form polarcontacts with the ligand (distance cutoff 3.5 Å) are in green, otherresidues are gray (in all panels, oxygens are colored red and nitrogensare blue). B. Same as panel A, except that the ligand is oriented withits amine facing out of the page. W109 is not shown. C. Packinginteractions between carazolol and all residues within 5 Å of theligand. View is from the extracellular side of the membrane. Carazololis shown as yellow spheres, receptor residues are shown as sticks withinvan der Waals dot surfaces. D. Model of (−)-isoproterenol (magentasticks) in the ligand binding pocket observed in the crystal structure.A model of the agonist with optimal bond lengths and angles was obtainedfrom the PRODRG server (Schuettelkopf, et al., Acta Crystallogr D BiolCrystallogr D60, 1355 (2004)), and the dihedral angles were adjusted tothe values observed in the homologous atoms of bound carazolol (16-22 inFIG. 24). The one remaining unaccounted dihedral in (−)-isoproterenolwas adjusted in order to place the catechol ring in the same plane asthe C₁₆-C₁₅-O₁₄ plane in carazolol. Residues known to specificallyinteract with agonists are shown as green sticks.

FIG. 26. Packing interactions in the β₂AR that are likely to bemodulated during the activation process. A. On the left, residuespreviously demonstrated to be CAMs (Rasmussen et al., Mol Pharmacol 56,175 (1999); Tao, et. al., Mol Endocrinol 14, 1272 (2000); Jensen et al.,J Biol Chem 276, 9279 (2001); Shi et al., J Biol Chem 277, 40989 (2002);Zuscik, et. al., (1998)) or UCMs (Strader et al., Proc Natl Acad Sci U SA 84, 4384 (1987); Chung, et al., J Biol Chem 263, 4052 (1988); Moro,et. al., J Biol Chem 269, 6651 (1994); Green, et. al., J Biol Chem 268,23116 (1993); Gabilondo et al., Proc Natl Acad Sci U S A 94, 12285(1997)) are shown as van der Waals spheres mapped onto a backbonecartoon of the β₂AR-T4L structure. On the right, residues that are foundwithin 4A of the CAMs Leu124^(3.43) and Leu272^(6.34) are shown asyellow spheres or dot surfaces. A vertical cross-section through thestructure illustrates that these surrounding residues connect the CAMson helices III and VI with the UCMs on helix VII through packinginteractions. B. In both β₂AR-T4L (blue) and rhodopsin (purple), anetwork of ordered water molecules is found at the interface between thetransmembrane helices at their cytoplasmic ends. C. Network of hydrogenbonding interactions between water molecules and β₂AR-T4L residues(sidechains as blue sticks), notably the UCMs on helix VII (orangecartoon).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods and compositions forgenerating crystal structures of membrane proteins that diffract toresolutions as low as 2 to 3 {acute over (Å)}ngstroms. In oneembodiment, the methods involve the crystallization of proteins in alipidic cubic phase, wherein the host lipid comprises an additive, e.g.,a sterol, such as cholesterol. The invention also provides thecrystallized membrane proteins themselves, wherein the crystallizedmembrane proteins include GPCRs or modified GPCRs. The crystallizedproteins can also include bound ligands, natural agonists, antagonists,and/or allosteric effectors. The invention additionally provides methodsof using the 3-dimensional structures of the proteins (obtained from thecrystals) to screen for novel ligands, drugs, and other useful moleculesthat affect the conformation and/or activity of the proteins in vitro orin vivo.

More specifically, the invention provides particular crystal forms ofGPCRs diffracting to high resolutions. GPCRs have been grouped into fiveclasses (Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based onsequence conservation, with class A GPCRs, including β₂AR, being thelargest and most studied. β₂AR agonists are used in the treatment ofasthma and preterm labor (DeLano, The PyMOL Molecular Graphics System(2002) on World Wide Web at pymol.org). The crystal forms provided bythe invention include several diffraction-quality class A GPCR crystals,including crystals comprising β2AR and crystals comprising the humanadenosine A_(2A) receptor.

The invention provides a three-dimensional structure of a human β₂ARprotein comprising a T4-lysozyme (T4L) in place of the thirdintracellular loop (“β₂AR-T4L”) that has been solved in the presence ofcarazolol (2-propanol, 1-(9H-carbazol-4-yloxy)-3-[(1-methylethyl)amino]at 2.4 Å resolution. Additional class A GPCR structures make it possibleto correlate sequence differences between GPCRs, e.g., between rhodopsinand β2AR, with empirically determined structural differences andextrapolate to other class A GPCRs. Highlighting interactions thatconstrain class A receptors into each of the two observed states allowsa more comprehensive analysis of structural divergence and, therefore,more accurate models. Furthermore, GPCR structures provide analternative signaling state on which to base homology models that willbe more relevant for virtual ligand screening and structure-based drugdesign (Bissantz, et. al, Proteins 50, 5 (2003); Gouldson et al.,Proteins 56, 67 (2004)).

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning as iscommonly understood by one of skill in the art to which this inventionbelongs.

As used herein, the term “binding site” or “binding pocket” refers to aregion of a protein that binds or interacts with a particular compound.

As used herein, the terms “binding” or “interaction” refers to acondition of proximity between a chemical entity, compound, or portionsthereof, with another chemical entity, compound or portion thereof. Theassociation or interaction can be non-covalent—wherein the juxtapositionis energetically favored by hydrogen bonding or van der Waals orelectrostatic interactions—or it can be covalent.

As used herein, the term “residue” refers to an amino acid that isjoined to another by a peptide bond. Residue is referred to herein todescribe both an amino acid and its position in a polypeptide sequence.

As used herein, the term “surface residue” refers to a residue locatedon a surface of a polypeptide. In contrast, a buried residue is aresidue that is not located on the surface of a polypeptide. A surfaceresidue usually includes a hydrophilic side chain. Operationally, asurface residue can be identified computationally from a structuralmodel of a polypeptide as a residue that contacts a sphere of hydrationrolled over the surface of the molecular structure. A surface residuealso can be identified experimentally through the use of deuteriumexchange studies, or accessibility to various labeling reagents such as,e.g., hydrophilic alkylating agents.

As used herein, the term “polypeptide” refers to a single linear chainof 2 or more amino acids. A protein is an example of a polypeptide.

As used herein, the term “homolog” refers to a gene related to a secondgene by descent from a common ancestral DNA sequence. The term, homolog,can apply to the relationship between genes separated by the event ofspeciation or to the relationship between genes separated by the eventof genetic duplication.

As used herein, the term “conservation” refers to a high degree ofsimilarity in the primary or secondary structure of molecules betweenhomologs. This similarity is thought to confer functional importance toa conserved region of the molecule. In reference to an individualresidue or amino acid, conservation is used to refer to a computedlikelihood of substitution or deletion based on comparison withhomologous molecules.

As used herein, the term “distance matrix” refers to the method used topresent the results of the calculation of an optimal pairwise alignmentscore. The matrix field (i,j) is the score assigned to the optimalalignment between two residues (up to a total of i by j residues) fromthe input sequences. Each entry is calculated from the top-leftneighboring entries by way of a recursive equation.

As used herein, the term “substitution matrix” refers to a matrix thatdefines scores for amino acid substitutions, reflecting the similarityof physicochemical properties, and observed substitution frequencies.These matrices are the foundation of statistical techniques for findingalignments.

As used herein, the term “pharmacophore” refers to an ensemble of stericand electronic features that is necessary to ensure the optimalsupramolecular interactions with a specific biological target structureand to trigger or block a biological response. A pharmacophore can beused to design one or more candidate compounds that comprise all or mostof the ensemble of steric and electronic features present in thepharmacophore and that are expected to bind to a site and trigger orblock a biological response.

As used herein, the term “G-protein coupled receptor” (or “GPCR”) refersto a member of a family of heterotrimeric guanine-nucleotide bindingprotein (“G-protein”) coupled receptors (Pierce, et al., Nat. Rev. Mol.Cell. Biol. 3:630 (2002)). GPCRs share a common structural signature ofseven membrane-spanning helices with an extra-cellular N terminus and anintracellular C terminus. The family has been grouped into at least fiveclasses (designated A, B, C, D, E, etc.; see, e.g., Fredriksson, et al.,Mol Pharmacol 63, 1256 (2003)) based on sequence conservation. When usedwithout a descriptive limitation, the term “a G-protein couple receptor”includes GPCRs with native amino acid sequences as well as geneticallyengineered or otherwise mutated GPCR proteins. Mutated GPCR proteinsinclude those comprising point mutations, truncations, insertedsequences or other chemical modifications, while retaining ligandbinding activity. One example of a GPCR referred to herein thatcomprises a point mutation is β2AR^(E122W). An example of a GPCRreferred to herein that comprises an inserted T4 lysozyme sequence isthe human A_(2a) adenosine receptor-T4L.

Adrenergic receptors in the class A or amine group are some of the mostthoroughly investigated GPCRs (Kobilka, Annu Rev Neurosci 15, 87 (1992);Caron, et al., Recent Prog Horm Res 48, 277 (1993); Strosberg, ProteinSci 2, 1198 (1993); Hein, et al., Trends Cardiovasc Med 7, 137 (1997);Rohrer, J Mol Med 76, 764 (1998); Xiang, et al., Adrenergic Receptors,267 (2006)), and are composed of two main subfamilies, α and β, whichdiffer in tissue localization and ligand specificity, as well as in Gprotein coupling and downstream effector mechanisms (Milligan, et al.,Biochem Pharmacol 48, 1059 (1994)). Some representative class Areceptors include the human A_(2A) adenosine receptor and the beta-2adrenergic receptor. The term “beta-2 adrenergic receptor” (or “β₂AR” or“β2AR”) refers to a class A GPCR that responds to diffusible hormonesand neurotransmitters and resides predominantly in smooth musclesthroughout the body. When used without a descriptive limitation, theterm “β2AR” includes β2ARs with native amino acid sequences as well asgenetically engineered or otherwise mutated β2AR proteins. Mutated β2ARproteins include those comprising point mutations, truncations, insertedsequences or other chemical modifications, while retaining ligandbinding activity. One example of a β2AR referred to herein thatcomprises a point mutation is β2AR^(E122W). An example of a β2ARreferred to herein that comprises an inserted T4 lysozyme sequence isthe human adenosine receptor β2AR^(E122W)-T4L.

The term “diffracts to a resolution of xx-yy Angstroms” means thatdiffraction data exceeding a predetermined signal to noise ratio can beobtained within the stated resolution range. In some embodiments, thatdiffraction data can be obtained using synchrotron radiation. Also, insome embodiments, that diffraction data can be obtained followingfreezing of the crystal in liquid nitrogen.

As used herein, the term “atomic co-ordinates” refers to a set ofthree-dimensional co-ordinates for atoms within a molecular structure.In one embodiment, atomic-co-ordinates are obtained using X-raycrystallography according to methods well-known to those of ordinarilyskill in the art of biophysics. Briefly described, X-ray diffractionpatterns can be obtained by diffracting X-rays off a crystal. Thediffraction data are used to calculate an electron density map of theunit cell comprising the crystal; said maps are used to establish thepositions of the atoms (i.e., the atomic co-ordinates) within the unitcell. Those of skill in the art understand that a set of structureco-ordinates determined by X-ray crystallography contains standarderrors. In other embodiments, atomic co-ordinates can be obtained usingother experimental biophysical structure determination methods that caninclude electron diffraction (also known as electron crystallography)and nuclear magnetic resonance (NMR) methods. In yet other embodiments,atomic co-ordinates can be obtained using molecular modeling tools whichcan be based on one or more of ab initio protein folding algorithms,energy minimization, and homology-based modeling. These techniques arewell known to persons of ordinary skill in the biophysical andbioinformatic arts, and are described in greater detail below.

Atomic co-ordinates for binding pockets, such as, e.g., the ligandbinding pocket of β2AR, and/or other agonist/antagonist binding sites ofthe present invention are intended to encompass those co-ordinates setout in the .pdb file (Appendix I; SEQ ID NOS 4-5, 1 and 6-9,respectively in order of appearance) incorporated into thisspecification, as well as co-ordinates that are substantiallyequivalent. Substantially equivalent co-ordinates are those that can berelated to a reference set of co-ordinates by transformation reflectingdifferences in the choice of origin or inter-axis angels for one or moreaxes used to define the coordinate system. Operationally, co-ordinatesare “substantially equivalent” when the structures represented by thoseco-ordinates can be superimposed in a manner such that root mean squaredeviations (RMSD) of atomic positions for the structures differs by lessthan a predetermined threshold. In some embodiments that threshold isless than about 5 Angstroms, or less than about 4 Angstroms, or lessthan about 3 Angstroms, or less than about 2 Angstroms, or less thanabout 1 Angstrom, or less than about 0.9 Angstrom, or less than about0.8 Angstrom, or less than about 0.7 Angstrom, or less than about 0.6Angstrom, or less than about 0.5 Angstrom, or less than about 0.4Angstrom, or less than about 0.3 Angstrom. Preferably, co-ordinates areconsidered “substantially equivalent” when the RMSD is less than about 1Angstrom. Methods for structure superpositioning and RMSD calculationsare well known to those of ordinary skill in the art, and can be carriedout using programs such as, e.g., the programs listed in Table 5 below.

Structural similarity can be inferred from, e.g., sequence similarity,which can be determined by one of ordinary skill through visualinspection and comparison of the sequences, or through the use ofwell-known alignment software programs such as CLUSTAL (Wilbur et al.,J. Proc. Natl. Acad. Sci. USA, 80, 726 730 (1983)) or CLUSTALW (Thompsonet al., Nucleic Acids Research, 22:4673 4680 (1994)) or BLAST®.(Altschul et al., J Mol. Biol., October 5; 215(3):403 10 (1990)), a setof similarity search programs designed to explore all of the availablesequence databases regardless of whether the query is protein or DNA.CLUSTAL W is available at the EMBL-EBI website(http://www.ebi.ac.uk/clustalw/); BLAST is available from the NationalCenter for Biotechnology website (http://www.ncbi.nlm.nih.gov/BLAST/). Aresidue within a first protein or nucleic acid sequence corresponds to aresidue within a second protein or nucleic acid sequence if the tworesidues occupy the same position when the first and second sequencesare aligned.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence co-ordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (NCBI web-site).

The term “sterol” refers to a subgroup of steroids with a hydroxyl groupat the 3-position of the A-ring. See Fahy E. Subramaniam S et al., “Acomprehensive classification system for lipids,” J. Lipid Res. 46(5):839-861 (2005)). Sterols are amphipathic lipids synthesized fromacetyl-coenzyme A via the HMG-CoA reductase pathway. The overallmolecule is quite flat. Sterols can include, e.g., cholesterol orcholesteryl hemisuccinate (“CHS”).

The term “atomic co-ordinates for residues” refers to co-ordinates forall atoms associated with a residue, or for some of the atoms such as,e.g., side chain atoms.

The term “atomic co-ordinates of a candidate compound” refers toco-ordinates for all atoms comprising the compound or a subset of atomscomprising the compound.

The term “characterizing a binding interaction” refers to characterizingany observable property of a first molecule and determining an whetherthere is a change in that observable property after contacting the firstmolecule with a second molecule under conditions in which said first andsecond molecules can potentially bind.

The term “antagonist” refers to molecules that bind to and block theactive site of a protein, but do not affect the equilibrium betweeninactive and active states. In contrast, an “agonist” is a ligand thatshifts the equilibrium to an active receptor state. An “inverse agonist”is a ligand that acts to reduce the basal activity of a receptor throughinteractions that shift the equilibrium to more of an inactive state.

Ballesteros-Weinstein numbering appears in the text and Figures assuperscripts to the protein numbering. Within each helix is a singlemost conserved residue among the class A GPCRs. This residue isdesignated X.50, where x is the number of the transmembrane helix. Allother residues on that helix are numbered relative to this conservedposition.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Method for Producing Diffraction Quality Crystals of Membrane Proteins

In one aspect, the present invention discloses a modified lipidic cubicmesophase method for crystallizing proteins (see, e.g., Cherezov et al.,Biophysical J., v. 83, 3393-3407 (2002)). The novel method describedherein yields diffraction quality crystals of membrane proteins and isparticularly useful for generating crystals of G-protein coupledreceptor proteins (“GPCRs”). The method has now been successfullyapplied to diverse members of this important family of proteins,yielding crystals that diffract to resolutions in the 2.5 {acute over(Å)} range. Among other advantages, this method allowsdiffraction-quality crystals of membrane proteins to be generated in theabsence of any stabilizing antibodies bound to the protein in thecrystal.

The LCP/sterol crystallization method described herein includes a stepof mixing a solution containing the protein of interest with a hostlipid or a host lipid mixture that includes a lipid additive. Given theteaching provided herein, one skilled in the art will recognize that avariety of host lipids may suffice for the generation of a cubicmesophase, e.g., hydrated monounsaturated monoacylglycerols such asmonoolein, monopalmitolein, and/or monovacennin. The host lipid1-monoolein is a preferred host lipid for certain applications of themethod. In embodiments utilizing a lipid mixture, a lipid additive thatis distinct from the host lipid is included, e.g., monounsaturatedmonoacylglycerols or other hydrophobic molecules known to interact withmembranes or membrane-associated proteins such as, 2-monoolein,phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid,dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoylphosphatidylcholine (“DOPC”), Asolectin, or a sterol (e.g., cholesterol,ergosterol, etc.). An example of a lipid mixture for GPCRcrystallization is one comprising cholesterol as lipid additive in aratio between 1 and 50% w/w relative to the host lipid, more preferablybetween 5 and 20%, and even more preferably between 8 and 12%. Theprotein mixture may include ligands of physiological interest and/orligands that stabilize the protein. In the case of GPCRs, the ligandsmay include various agonists and antagonists known to the artisan,including well-known agonists such as carazolol (an inverse agonist),timolol, and other molecules including, without limitation, Examples ofligands, include but are not limited to carazolol, light and olfactorystimulatory molecules; adenosine, bombesin, bradykinin, endothelin,y-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins,neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins,vasoactive intestinal polypeptide family, and vasopressin; biogenicamines (e.g., dopamine, epinephrine and norepinephrine, histamine,glutamate (metabotropic effect), glucagon, acetylcholine (muscariniceffect), and serotonin); chemokines; lipid mediators of inflammation(e.g., prostaglandins and prostanoids, platelet activating factor, andleukotrienes); and peptide hormones (e.g., calcitonin, C5aanaphylatoxin, follicle stimulating hormone (FSH),gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropinreleasing hormone (TRH), and oxytocin).

A typical concentration of protein in the protein mixture is 25-75mgs/ml but this concentration may vary according to protein identity andpurification methods. As will be recognized by the skilled artisan, theconcentration must be high enough to result in a degree of insolubilitysufficient for nucleation to occur after a precipitation solution iscombined with the protein-laden lipid solution; on the other hand,concentrations of protein that are too high may prevent the orderlygrowth of high-quality crystals.

The lipid mixture is preferably combined with the protein mixture andhomogenized, e.g., using a syringe mixer, spontaneously yielding ahomogenous cubic phase. Typically, the lipid mixture is added to theprotein solution at a ratio of 1:1, 3:2, 4:2 w/w lipid:protein, but thisratio may be varied by the skilled artisan as desired, depending onvarious parameters, e.g., the concentration of protein in the proteinmixture. The protein-laden lipidic cubic phase preparation thus obtainedis then combined with precipitation solution (also referred to ascrystallization solution) on or in an appropriate surface or container,e.g., a glass sandwich plate with wells where the mixed solutions canincubate while crystallization occurs. A typical volume of theprotein-laden lipidic cubic phase used in the method is between 10 and100 nL, with 40 to 60 nL preferred in certain embodiments. A typicalvolume of precipitation solution is 20 to 100 times greater, e.g., for a20 nL volume of protein-laden lipidic cubic phase, approximately 1 μL ofprecipitation solution would be added to initiate crystallization.

The precipitation solution used in the crystallization method is anappropriately buffered solution (i.e., buffered to approximate thephysiological conditions of the native protein) comprising polyethyleneglycol, a salt, and optionally a small soluble molecule such as analcohol.

With respect to the polyethylene glycol in the precipitation solution,useful PEG molecules include PEG 300, PEG 400, PEG 550, PEG 550mme, PEG1000, and PEG 1500, as well as other PEG molecules with averagemolecular weights less than 2000. In certain embodiments, larger averagemolecular weight PEG molecules (up to 20,000) or modified PEG moleculesmay be preferred. In some embodiments, the PEG or modified PEG has anaverage molecular weight of 400. Examples of modified PEG include butare not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate,PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryllaurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kerneloil, PEG hydrogenated castor oil, PEG castor oil, PEG corn oil, PEGcaprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEGcholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEGsorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenolseries, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHSEster and methoxypoly(ethylene glycol) (mPEG).

PEG may be present in the crystallization solution in concentrationsbetween 10-60% v/v, and most typically between 20-40% v/v. The preferredconcentration will vary depending on the average molecular weight of PEGutilized, i.e., 10-60% v/v of PEG will be preferred for PEG ≦1000whereas 10-30% w/v will be preferred for PEG >1000 (larger averagemolecular weight PEG formulations are described in % w/v rather than %v/v).

With respect to the salt used in the method, an optimum cation canusually be found for a given crystal. Both sodium and lithium sulfatehave proven useful for obtaining high resolution proteins of GPCRs.Again, the concentrations may be varied up to 1M, with lowerconcentrations of approximately 50-200 mM typically preferred. Otherorganic salts, e.g., citrate, malonate, tartrate, formate and acetate,may also be screened for their effects on crystal formation. In certainembodiments, the precipitation solution additionally comprises a smallorganic molecule such as an alcohol, a diol or a triol, e.g., ahexanediol, a butanediol, or derivative thereof. These molecules may bepresent in the precipitation solution in various concentrations, asappropriate, but typically in the range of 1-20% v/v, more typically inthe 5-10% v/v range. In certain embodiments, preferred combinations oflipid additives (in the protein-laden lipidic cubic phase mixture) andsmall molecules (in the precipitation solution) yield optimal results.Examples of such combinations include 1,4-butanediol in combination withDOPE or cholesterol, and 2,6-hexanediol in combination with cholesterol.

In optimizing the conditions from micro-crystals to larger crystals fora given system (e.g., a protein/ligand system), the choice andconcentration of a specific sterol(s) and specific lipid(s), as well thepH, buffer system, salt, and salt concentration may be varied, as inother types of crystallization formats. As noted above, small organicadditives, especially alcohols and diols such as 1,4 butanediol, 1,6hexanediol, etc., can be particularly useful in generating largediffraction quality crystals. Also, due to the membranefluidity-altering properties of cholesterol and other sterols, steroland precipitant concentration should be treated as dependent variables.For example, increasing concentrations of cholesterol in monoolein serveto rigidify the membranes, potentially slowing diffusion of the membraneprotein within the lipid matrix. Conversely, increasing concentrationsof PEG 400 swell the cubic phase, thereby increasing the latticeparameter of the matrix and speeding diffusion within the lipid. Theformer scenario would slow the rate of crystallization while the latterwould increase the rate. The two effects should therefore be balancedfor optimal nucleation and also for optimal growth of large,well-ordered crystals that diffract to a high resolution.

The mixing of the protein-laden lipidic cubic phase solution and theprecipitation solution typically occurs at room temperatures. Afterset-up, the plates containing the mixed crystallization solutions can bemonitored as often as desired for the appearance of crystal growth. Oneskilled in the art will recognize that further optimization of theseconditions may be desirable, for example, to maximize the size andnumber of diffraction quality crystals that are obtained. In makingdeterminations as to the preferred molecules and conditions forcrystallization, the skilled artisan may rely on well-known phasediagrams and other previously determined physical constants, in additionto the novel methodology and Examples described herein. For certainlipid mixtures, pre-screening their phase behavior by microscopevisualization and/or by X-ray prior to being mixed with the proteinsolution may facilitate the process of optimization. An in mesocrystallization robot and automatic imager combined with multiple96-well optimization screens can be used to run thousands of trials in arelatively facile manner.

It also possible to achieve additional stabilization of proteins andimprove the yield of diffraction-quality crystals using the LCP/sterolmethod described herein by modification of the protein. For example, anunstable region of the protein may be replaced or stabilized byincorporation of a portion of a stable protein, e.g., a T4 lysozyme,whose structure is previously known but which does not (when fused)significantly affect the biochemical activity of the protein ofinterest. For example, the ECL2 and ECL3 regions of a β2AR can bestabilized by such modifications, as described herein (Examples 3 and4). Other modifications include one or more point mutations that do notsignificantly alter the properties of the protein of interest except toincrease its stability and/or tendency to crystallize well. For example,β2AR^((E122W)) comprises an E122W point mutation and yields crystalswith the LPC/sterol method. Analogous residues in other GPCRs could bemodified in the same way. One advantage of the LCP method applied toboth modified and unmodified proteins, as noted above, is that it allows(but does not preclude) the crystallization of proteins in the absenceof heterologous proteins, such as antibodies, that may not be ofinterest to the crystallographer.

Method of Ligand Screening by Lipidic Cubic Phase Crystallization

Aspects of the lipidic cubic phase crystallization methodology describedabove can be modified for the purpose of determining low affinity lipidbinding sites within integral membrane proteins throughco-crystallization trials within a lipidic cubic phase matrix. In thismethod various lipids of different composition are incorporated at avariety of concentrations into monoolein, wherein the monoolein issolubilized in chloroform or heated to its fluid isotropic phase.Crystal growth is then assessed by visual inspections and diffractiondata collected on any crystalline material within the experiment.Because the lipid is low affinity the method requires an environmentconducive for free exchange of lipid from annular to non-annular proteinbinding sites. The presence of interpretable electron density notassociated with crystal packing interfaces allows the inference ofspecific binding sites for a particular lipid within the context of themembrane protein in a membrane environment. Because the binding occurswithin a membrane the complicating factor of detergent partitioning iseliminated and thermodynamics of association are more realistic. Themethod thus allows one to characterize in detail previously inaccessibleregions of membrane proteins, as well as describe and exploit bindinginteractions that might otherwise remain undetected. Furthermore, thetechnique can be applied to ligand binding studies where the ligandoccupies a site on the membrane protein that is juxtaposed to the lipidplane relying on partitioning into the aqueous phase to enablesaturation of the site. This limits the exposure of the protein to harshorganic co-solvents and may also find utility for soluble proteins thatcrystallize within the lipidic cubic phase solvent channels.

By wan of example, existing crystallization conditions for a protein canbe utilized as a starting point for screening novel ligands to theβ2-adrenergic receptor. In the first instance, cholesterol solubilizedin chloroform can be incorporated into chloroform-solubilized monooleinat weight ratio of 10%. After drying and desiccating the mixture,protein at 30-80 mg/mL can incorporated at 2/3 volume ratio and used forcrystallization trials. A similar protocol was used for other lipid likemolecules, including cholesteryl hemisuccinate and a variety of othercholesterol analogs. In each case protein was incorporated into theresulting mixture and screened for crystallization. Binding of the novelligand to the receptor is indicated by diffraction quality crystals andultimately by three-dimensional structural data. By incorporatinganalogues of cholesterol we are able to map out the bindingspecificities based on the unique structural features of theirrespective sterol rings and polar moieties and, if their incorporationled to diffraction quality crystals, the interactions between theprotein and cholesterol analogue are determined.

This method of ligand screening is not limited to lipid-like molecules,as we can use the lipidic cubic phase as a host for other highlyhydrophobic molecules that act at orthosteric binding sites. One problemwith structure based or fragment based design of novel ligands is thehydrophobicity often associated with potential drug leads. This is aproblem in aqueous based crystallization schemes because the solubilityof the ligand is often less than 1 mM and unless there is a slow offrate from the protein of interest the binding site will be in a liganddepleted state at crystallization conditions which often involve proteinconcentrations between 0.5 and 1 mM. One can attempt to co-solubilizethe hydrophobic ligand in aqueous miscible organic solvent such asdimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). However, theseoften interfere with the stability or crystallization properties of theprotein and their usefulness is not general. Therefore, this methodallows one to incorporate the hydrophobic ligand directly into thelipidic cubic phase where its accessibility to the protein will belimited by the partitioning between the lipid and aqueous phase and/orthe accessibility of the binding.

Additional guidance relating to these methods is provided by the workingand prophetic examples of protein crystallization presented herein.

The Crystal Structure of Human β2AR Bound to Carazolol and Uses Thereof

G-protein coupled receptors are cell surface receptors that indirectlytransduce extracellular signals to downstream effectors, e.g.,intracellular signaling proteins, enzymes, or channels. G-proteincoupled receptor membrane proteins are grouped into one of at least 6classes (i.e., A, B, C, D, E, and F). An example of a mammalianG-protein coupled receptor is the β2A receptor, a receptor in the ClassA subfamily of GPCRs.

Class A GPCRs function in a variety of physiological processes such asvasodilation, bronchodilation, neurotransmitter signaling, stimulationof endocrine secretions, gut peristalsis, development, mitogenesis, cellproliferation, cell migration, immune system function, and oncogenesis.Accordingly, class A GPCRs can be used as screening targets to identifymodulators of these processes which can then function to amelioratediseases associated with these processes, e.g., cancer and autoimmunity.

The 2.4 Angstrom structure of β2AR bound to carazolol, described herein(PDB coordinates appear in Appendix I; SEQ ID NOS 4-5, 1 and 6-9,respectively in order of appearance) can be used as a model forrationally designing pharmacophore and/or candidate compounds, either denovo or by modification of known compounds. As noted below, the multipleligand binding sites in this structure include amino acids that arehighly conserved across a large number of class A G protein coupledreceptors (GPCRs) indicating that the 2.4 Angstrom structure of β2AR canbe used for the rational designing of ligands (e.g., therapeuticcompounds) that bind to this receptor and others. Pharmacophore andcandidate compounds identified through the use of the crystal structureco-ordinates will have utility as pharmaceuticals due to their abilityto alter the structure and/or binding properties of β2AR. Pharmacophoresand candidate compounds can be determined according to any method knownin the art, including the methods described in U.S. Pat. No. 5,888,738to Hendry, and the methods described in U.S. Pat. No. 5,856,116 toWilson et al. the disclosures of which both are incorporated byreference in their entirety for all purposes.

The structure data provided herein can be used in conjunction withcomputer-modeling techniques to develop models of sites on the humanβ2AR or related GPCRs selected by analysis of the crystal structuredata. The site models characterize the three-dimensional topography ofsite surface, as well as factors including van der Waals contacts,electrostatic interactions, and hydrogen-bonding opportunities. Computersimulation techniques can be used to map interaction positions forfunctional groups including protons, hydroxyl groups, amine groups,divalent cations, aromatic and aliphatic functional groups, amidegroups, alcohol groups, etc. that are designed to interact with themodel site. These groups can be designed into a pharmacophore orcandidate compound with the expectation that the candidate compound willspecifically bind to the site. Pharmacophore design thus involves aconsideration of the ability of the candidate compounds falling withinthe pharmacophore to interact with a site through any or all of theavailable types of chemical interactions, including hydrogen bonding,van der Waals, electrostatic, and covalent interactions, although, ingeneral, and preferably, pharmacophores interact with a site throughnon-covalent mechanisms.

The ability of a pharmacophore or candidate compound to bind to thehuman β2AR can be analyzed prior to actual synthesis using computermodeling techniques. Only those candidates that are indicated bycomputer modeling to bind the target with sufficient binding energy(i.e., binding energy corresponding to a dissociation constant with thetarget on the order of 10⁻² M or tighter) can be synthesized and testedfor their ability to bind to the human β2AR using binding assays orfunctional assays known to those of skill in the art. The computationalevaluation step thus avoids the unnecessary synthesis of compounds thatare unlikely to bind β2AR or one or more of its constitutive bindingsites, or the related binding sites of another GPCR with adequateaffinity.

A human β2AR or candidate compound(s) can be computationally evaluatedand designed by means of a series of steps in which chemical entities orfragments are screened and selected for their ability to associate withindividual binding target sites on β2AR or binding site thereof,including, but not limited to a binding pocket of the human β2AR. Oneskilled in the art can use one of several methods to screen chemicalentities or fragments for their ability to associate with one or more ofthese human β2AR binding sites. For example, increased affinity andspecificity may be designed into caffeine and other xanthine moleculesby combining interactions with both xanthine and non-xanthine bindingsites.

The process can begin by visual inspection of, for example a target siteon a computer screen, based on the human β2AR co-ordinates, or a subsetof those co-ordinates (e.g., binding pocket residues V117, T118, F193,Y199, A200, W286, F289, F290, and Y316), as set forth in Appendix I (SEQID NOS 4-5, 1 and 6-9, respectively in order of appearance). Selectedfragments or chemical entities can then be positioned in a variety oforientations or “docked” within a target site of the human β2AR asdefined from analysis of the crystal structure data. Docking can beaccomplished using software such as Quanta (Molecular Simulations, Inc.,San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed byenergy minimization and molecular dynamics with standard molecularmechanics forcefields such as CHARMM (Molecular Simulations, Inc., SanDiego, Calif.), ICM (Molsoft, San Diego, Calif.), and AMBER (Universityof California, San Francisco).

Specialized computer programs can also assist in the process ofselecting fragments or chemical entities. These include but are notlimited to: GRID (Goodford, P. J., “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules,” J. Med. Chem., 28, pp. 849 857 (1985)); GRIDis available from Oxford University, Oxford, UK; MCSS (Miranker, A. andM. Karplus, “Functionality Maps of Binding Sites: A Multiple CopySimultaneous Search Method,” Proteins: Structure, Function and Genetics,11, pp. 29 34 (1991)); MCSS is available from Molecular Simulations,Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S, and A. J. Olsen,“Automated Docking of Substrates to Proteins by Simulated Annealing,”Proteins: Structure, Function, and Genetics, 8, pp. 195 202 (1990));AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.;DOCK (Kuntz, I. D., et al. “A Geometric Approach to Macromolecule-LigandInteractions,” J. Mol. Biol., 161, pp. 269 288 (1982)); DOCK isavailable from University of California, San Francisco, Calif.; CERIUSII (available from Molecular Simulations, Inc., San Diego, Calif.); andFlexx (Raret, et al. J. Mol. Biol. 261, pp. 470 489 (1996)).

After selecting suitable chemical entities or fragments, they can beassembled into a single compound. Assembly can proceed by visualinspection of the relationship of the fragments to each other on athree-dimensional image of the fragments in relation to the human β2ARor its binding sites or those of a related GPCR receptor structure orportion thereof displayed on a computer screen. Visual inspection can befollowed by manual model building using software such as the Quanta orSybyl programs described above.

Software programs also can be used to aid one skilled in the art inconnecting the individual chemical entities or fragments. These include,but are not limited to CAVEAT (Bartlett, P. A., et al. “CAVEAT: AProgram to Facilitate the Structure-Derived Design of BiologicallyActive Molecules” In “Molecular Recognition in Chemical and BiologicalProblems,” Special Publ, Royal Chem. Soc., 78, pp. 182 196 (1989));CAVEAT is available from the University of California, Berkeley, Calif.;3D Database systems such as MACCS-3D (MDL Information Systems, SanLeandro, Calif.); this area is reviewed in Martin, Y. C., “3D DatabaseSearching in Drug Design,” J. Med. Chem., 35:2145 2154 (1992)); and HOOK(available from Molecular Simulations Inc., San Diego, Calif.).

As an alternative to building candidate pharmacophores or candidatecompounds up from individual fragments or chemical entities, they can bedesigned de novo using the structure of the β2AR, its constituent ligandbinding pocket, or the homologous cavities in a related GPCR,optionally, including information from co-factor(s) or known activatorsor inhibitor(s) that bind to the target site. De novo design can beimplemented by programs including, but not limited to LUDI (Bohm, H. J.,“The Computer Program LUDI: A New Method for the De Novo Design ofEnzyme Inhibitors,” J. Comp. Aid. Molec. Design, 6, pp. 61 78 (1992));LUDI is available from Molecular Simulations, Inc., San Diego, Calif.;LEGEND (Nishibata, Y., and Itai, A., Tetrahedron 47, p. 8985 (1991);LEGEND is available from Molecular Simulations, San Diego, Calif.; andLeapFrog (available from Tripos Associates, St. Louis, Mo.).

The functional effects of known β2AR also can be altered through the useof the molecular modeling and design techniques described herein. Thiscan be carried out by docking the structure of the known ligand on ahuman A_(2A) adenosine receptor or a model structure of one or morebinding sites of the human β2AR (e.g., the binding pocket describedherein) and modifying the shape and charge distribution of the ligand orprotein model structure to optimize the binding interactions between theligand and protein. The modified structure can be synthesized orobtained from a library of compounds and tested for its binding affinityand/or effect on ribosome function. Of course, where the crystalstructure of a complex between a human β2AR (or subunit thereof) and aligand is known, comparisons between said complex and the structures ofthe present invention can be made to gain additional information aboutalterations in human β2AR conformation that occur upon ligand binding.This information can be used in design of optimized ligands. Compoundsthat interfere or activate human β2AR function (e.g., by interactingwith a binding pocket) are especially well suited for the docking,co-crystallization, and optimization applications of the presentinvention.

Additional molecular modeling techniques also can be employed inaccordance with the invention. See, e.g., Cohen, N. C., et al.“Molecular Modeling Software and Methods for Medicinal Chemistry,” J.Med. Chem., 33, pp. 883 894 (1990); Hubbard, Roderick E., “Can drugs bedesigned?” Curr. Opin. Biotechnol. 8, pp. 696 700 (1997); and Afshar, etal. “Structure-Based and Combinatorial Search for New RNA-BindingDrugs,” Curr. Opin. Biotechnol. 10, pp. 59 63 (1999).

Following pharmacophore or candidate compound design or selectionaccording to any of the above methods or other methods known to oneskilled in the art, the efficiency with which a candidate compoundfalling within the pharmacophore definition binds to the human β2AR orits ligand binding site, or alternatively binds to a related GPCR orhomologous portions thereof, can be tested and optimized usingcomputational evaluation. A candidate compound can be optimized, e.g.,so that in its bound state it would preferably lack repulsiveelectrostatic interaction with the target site. These repulsiveelectrostatic interactions include repulsive charge-charge,dipole-dipole, and charge-dipole interactions. It is preferred that thesum of all electrostatic interactions between the candidate compound andthe human β2AR, including its ligand binding site when the candidatecompound is bound to the target make a neutral or favorable contributionto the binding enthalpy or free energy.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include, but are not limited to Gaussian 92,revision C (Frisch, M. J., Gaussian, Inc., Pittsburgh, Pa. (1992));AMBER, version 4.0 (Kollman, P. A., University of California at SanFrancisco, (1994)); QUANTA/CHARMM (Molecular Simulations, Inc., SanDiego, Calif. (1994)); and Insight II/Discover (Biosym TechnologiesInc., San Diego, Calif. (1994)). These programs can be run, using, e.g.,a Silicon Graphics workstation, Indigo, 02-R10000 or IBM RISC/6000workstation model 550. Other hardware and software combinations can beused to carry out the above described functions, and are known to thoseof skill in the art. In general, the methods described herein,particularly computer-implemented methods, comprise a step of recordingor storing data onto a medium, wherein the medium can include acomputer-readable medium. Additionally, or alternatively, the methodscomprise a step of reporting or communicating the data to a user ofinterest, e.g., an operator of the device and/or computer that isemployed in the method; or the computer can perform an additional usefultask, e.g., alert the operator of the computer that a function has beencompleted, upon completing one or more determining steps of the method.

Once a pharmacophore or candidate compound has been optimally selectedor designed, as described above, substitutions can then be made in someof its atoms or side groups to improve or modify its binding properties.Generally, initial substitutions are conservative in that thereplacement group will have approximately the same size, shape,hydrophobicity and charge as the original group. Components known in theart to alter conformation should be avoided in making substitutions.Substituted candidates can be analyzed for efficiency of fit to thehuman β2AR (or one or more binding sites of the human β2AR) using thesame methods described above.

Assays

Any one of a number of assays of function known to those of skill in theart can be used to determine the biological activity of candidatecompounds.

Candidate compound interaction with the human β2AR (or one or morebinding sites of human β2AR) or to a related GPCR or portion thereof canbe evaluated using direct binding assays including filter bindingassays, such as are known to those skilled in the art. Binding assayscan be modified to evaluate candidate compounds that competitivelyinhibit the binding of, e.g., known human β2AR binding compoundsincluding xanthine and xanthine-based compounds such as theophylline,theobromine and caffeine. These and other assays are described inInternational Publication WO 00/69391, the entire disclosure of which isincorporated by reference in its entirety for all purposes. Methods ofassaying for modulators of ligand binding and signal transductioninclude in vitro ligand binding assays using GPCRs, such as human β2AR(or one or more binding sites selected from the binding pockets I, IIand III of the human β2AR), portions thereof such as the extracellulardomain, or chimeric proteins comprising one or more domains of a GPCR,oocyte GPCR expression or tissue culture cell GPCR expression, eithernaturally occurring or recombinant; membrane expression of a GPCR,either naturally occurring or recombinant; tissue expression of a GPCR;expression of a GPCR in a transgenic animal, etc.

As noted above, GPCRs and their alleles and polymorphic variants areG-protein coupled receptors that participate in signal transduction andare associated with cellular function in a variety of cells, e.g.,neurons, immune system cells, kidney, liver, colon, adipose, and othercells. The activity of GPCR polypeptides can be assessed using a varietyof in vitro and in vivo assays to determine functional, chemical, andphysical effects, e.g., measuring ligand binding, (e.g., radioactiveligand binding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, orCa²⁺), ion flux, phosphorylation levels, transcription levels,neurotransmitter levels, and the like. Such assays can be used to testfor inhibitors and activators of a GPCR. In particular, the assays canbe used to test for compounds that modulate natural ligand-induced GPCRactivity, for example, by modulating the binding of the natural ligandto the receptor and/or by modulating the ability of the natural ligandto activate the receptor. Typically in such assays, the test compound iscontacted with the GPCR in the presence of the natural ligand. Thenatural ligand can be added to the assay before, after, or concurrentlywith the test compound. The results of the assay, for example, the levelof binding, calcium mobilization, etc. is then compared to the level ina control assay that comprises the GPCR and natural ligand in theabsence of the test compound.

Screening assays of the invention are used to identify modulators thatcan be used as therapeutic agents, e.g., antagonists of GPCR activity.For example, carazolol is a known high-affinity inverse agonist of humanβ2AR.

The effects of test compounds upon the function of the GPCR polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the GPCRs and naturalligand-mediated GPCR activity. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as Ca²⁺,IP₃ or cAMP.

For a general review of GPCR signal transduction and methods of assayingsignal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238(1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27 (1991);Bourne et al., Nature 348:125-32 (1990); Pitcher et al., Annu. Rev.Biochem. 67:653-92 (1998).

Modulators of GPCR activity are tested using GPCR polypeptides, eitherrecombinant or naturally occurring. The protein can be isolated,expressed in a cell, expressed in a membrane derived from a cell,expressed in tissue or in an animal, either recombinant or naturallyoccurring. For example, neurons, cells of the immune system, adipocytes,kidney cells, transformed cells, or membranes can be used. Modulation istested using one of the in vitro or in vivo assays described herein orothers as generally known in the art. Signal transduction can also beexamined in vitro with soluble or solid state reactions, using achimeric molecule such as an extracellular domain of a receptorcovalently linked to a heterologous signal transduction domain, or aheterologous extracellular domain covalently linked to the transmembraneand or cytoplasmic domain of a receptor. Furthermore, ligand-bindingdomains of the protein of interest can be used in vitro in soluble orsolid state reactions to assay for ligand binding.

Ligand binding to a human β2AR (or one or more binding sites thereof) ora chimeric protein derivative can be tested in a number of formats. Forexample, binding can be performed in solution, in a bilayer membrane,attached to a solid phase, in a lipid monolayer, or in vesicles.Typically, in an assay of the invention, the binding of the naturalligand to its receptor is measured in the presence of a candidatemodulator. Alternatively, the binding of the candidate modulator can bemeasured in the presence of the natural ligand. Often, competitive assaythat measure the ability of a compound to compete with binding of thenatural ligand to the receptor are used. Binding can be measured byassessing GPCR activity or by other assays: binding can be tested bymeasuring e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape)changes, or changes in chromatographic or solubility properties.

Receptor-G-protein interactions can also be used to assay formodulators. For example, in the absence of GTP, binding of an activatorsuch as the natural ligand will lead to the formation of a tight complexof a G protein (all three subunits) with the receptor. This complex canbe detected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors. For example, a ligand can be added tothe human β2AR and G protein in the absence of GTP to form a tightcomplex. Inhibitors can be identified by looking at dissociation of thereceptor-G protein complex. In the presence of GTP, release of the alphasubunit of the G protein from the other two G protein subunits serves asa criterion of activation.

An activated or inhibited G-protein will in turn alter the properties ofdownstream effectors such as proteins, enzymes, and channels. Theclassic examples are the activation of cGMP phosphodiesterase bytransducin in the visual system, adenylate cyclase by the stimulatoryG-protein, phospholipase C by G_(q) and other cognate G proteins, andmodulation of diverse channels by Gi and other G proteins. Downstreamconsequences such as generation of diacyl glycerol and IP₃ byphospholipase C, and in turn, for calcium mobilization e.g., by IP₃ canalso be examined. Thus, modulators can be evaluated for the ability tostimulate or inhibit ligand-mediated downstream effects. In otherexamples, the ability of a modulator to activate a GPCR expressed inadipocytes in comparison to the ability of a natural ligand, can bedetermined using assays such as lipolysis (see, e.g., WO01/61359).

Activated GPCRs become substrates for kinases that phosphorylate theC-terminal tail of the receptor (and possibly other sites as well).Thus, activators will promote the transfer of ³²P from gamma-labeled GTPto the receptor, which can be assayed with a scintillation counter. Thephosphorylation of the C-terminal tail will promote the binding ofarrestin-like proteins and will interfere with the binding ofG-proteins. The kinase/arrestin pathway plays a key role in thedesensitization of many GPCR receptors. Modulators can therefore also beidentified using assays involving beta-arrestin recruitment.Beta-arrestin serves as a regulatory protein that is distributedthroughout the cytoplasm in unactivated cells. Ligand binding to anappropriate GPCR is associated with redistribution of beta-arrestin fromthe cytoplasm to the cell surface, where it associates with the GPCR.Thus, receptor activation and the effect of candidate modulators onligand-induced receptor activation, can be assessed by monitoringbeta-arrestin recruitment to the cell surface. This is frequentlyperformed by transfecting a labeled beta-arrestin fusion protein (e.g.,beta-arrestin-green fluorescent protein (GFP)) into cells and monitoringits distribution using confocal microscopy (see, e.g., Groarke et al.,J. Biol. Chem. 274(33):23263-69 (1999)).

Receptor internalization assays can also be used to assess receptorfunction. Upon ligand binding, the G-protein coupled receptor—ligandcomplex is internalized from the plasma membrane by a clathrin-coatedvesicular endocytic process; internalization motifs on the receptorsbind to adaptor protein complexes and mediate the recruitment of theactivated receptors into clathrin-coated pits and vesicles. Because onlyactivated receptors are internalized, it is possible to detectligand-receptor binding by determining the amount of internalizedreceptor. In one assay format, cells are transiently transfected withradiolabeled receptor and incubated for an appropriate period of time toallow for ligand binding and receptor internalization. Thereafter,surface-bound radioactivity is removed by washing with an acid solution,the cells are solubilized, and the amount of internalized radioactivityis calculated as a percentage of ligand binding. See, e.g., Vrecl etal., Mol. Endocrinol. 12:1818-29 (1988) and Conway et al., J. CellPhysiol. 189(3):341-55 (2001). In addition, receptor internalizationapproaches have allowed real-time optical measurements of GPCRinteractions with other cellular components in living cells (see, e.g.,Barak et al., Mol. Pharmacol. 51(2)177-84 (1997)). Modulators can beidentified by comparing receptor internalization levels in control cellsand cells contacted with candidate compounds. For example, candidatemodulators the human β2AR are assayed by examining their effects onreceptor internalization upon binding of the natural ligand.

Another technology that can be used to evaluate GPCR-proteininteractions in living cells involves bioluminescence resonance energytransfer (BRET). A detailed discussion regarding BRET can be found inKroeger et al., J. Biol. Chem., 276(16):12736-43 (2001).

Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([³⁵S]GTPγS)binding to G-proteins can also be used as an assay for evaluatingmodulators of GPCRs. [³⁵S]GTPγS is a radiolabeled GTP analog that has ahigh affinity for all types of G-proteins, is available with a highspecific activity and, although unstable in the unbound form, is nothydrolyzed when bound to the G-protein. Thus, it is possible toquantitatively assess ligand-bound receptor by comparing stimulatedversus unstimulated [³⁵S]GTPγS binding utilizing, for example, a liquidscintillation counter. Inhibitors of the receptor-ligand interactionswould result in decreased [³⁵S]GTPγS binding. Descriptions of [³⁵S]GTPγSbinding assays are provided in Traynor and Nahorski, Mol. Pharmacol.47(4):848-54 (1995) and Bohn et al., Nature 408:720-23 (2000).

The ability of modulators to affect ligand-induced ion flux can also bedetermined. Ion flux can be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a GPCR. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., Pflügers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales &Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth.25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors and thenatural ligands disclosed herein as negative or positive controls toassess activity of tested compounds. In assays for identifyingmodulatory compounds (e.g., agonists, antagonists), changes in the levelof ions in the cytoplasm or membrane voltage are monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. Amongthe ion-sensitive indicators and voltage probes that can be employed arethose disclosed in the Molecular Probes 1997 Catalog. For G-proteincoupled receptors, promiscuous G-proteins such as Gα15 and Gα16 can beused in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of awide range of receptors to signal transduction pathways in heterologouscells.

Receptor activation by ligand binding typically initiates subsequentintracellular events, e.g., increases in second messengers such as IP₃,which releases intracellular stores of calcium ions. Activation of someG-protein coupled receptors stimulates the formation of inositoltriphosphate (IP₃) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP₃in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP₃ can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors canexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it can be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated by ligand binding, result in a change in the level ofintracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating orinhibiting downstream effectors such as adenylate cyclase. There arecyclic nucleotide-gated ion channels, e.g., rod photoreceptor cellchannels and olfactory neuron channels that are permeable to cationsupon activation by binding of cAMP or cGMP (see, e.g., Altenhofen etal., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) and Dhallan etal., Nature 347:184-187 (1990)). In cases where activation of thereceptor results in a decrease in cyclic nucleotide levels, it can bepreferable to expose the cells to agents that increase intracellularcyclic nucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay. Cells for thistype of assay can be made by co-transfection of a host cell with DNAencoding a cyclic nucleotide-gated ion channel, GPCR phosphatase and DNAencoding a receptor (e.g., certain glutamate receptors, muscarinicacetylcholine receptors, dopamine receptors, serotonin receptors, andthe like), which, when activated, causes a change in cyclic nucleotidelevels in the cytoplasm.

In one embodiment, changes in intracellular cAMP or cGMP can be measuredusing immunoassays. The method described in Offermanns & Simon, J. Biol.Chem. 270:15175-15180 (1995) can be used to determine the level of cAMP.Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell andMol. Biol., 11:159-164 (1994) can be used to determine the level ofcGMP. Further, an assay kit for measuring cAMP and/or cGMP is describedin U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates areseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which can or can not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on ligand-induced signal transduction. Ahost cell containing the protein of interest is contacted with a testcompound in the presence of the natural ligand for a sufficient time toeffect any interactions, and then the level of gene expression ismeasured. The amount of time to effect such interactions can beempirically determined, such as by running a time course and measuringthe level of transcription as a function of time. The amount oftranscription can be measured by using any method known to those ofskill in the art to be suitable. For example, mRNA expression of theprotein of interest can be detected using northern blots or theirpolypeptide products can be identified using immunoassays.Alternatively, transcription based assays using reporter genes can beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,beta-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it can be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell can be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

Samples that are treated-with a potential GPCR inhibitor or activatorare compared to control samples comprising the natural ligand withoutthe test compound to examine the extent of modulation. Control samples(untreated with activators or inhibitors) are assigned a relative GPCRactivity value of 100. Inhibition of a GPCR is achieved when the GPCRactivity value relative to the control is about 90%, optionally 50%,optionally 25-0%. Activation of a GPCR is achieved when the GPCRactivity value relative to the control is 110%, optionally 150%,200-500%, or 1000-2000%.

In one embodiment the invention provides soluble assays using moleculessuch as a domain, e.g., a ligand binding domain, an extracellulardomain, a transmembrane domain (e.g., one comprising seven transmembraneregions and cytosolic loops), the transmembrane domain and a cytoplasmicdomain, an active site, a subunit association region, etc.; a domainthat is covalently linked to a heterologous protein to create a chimericmolecule; a GPCR; or a cell or tissue expressing a GPCR, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where the domain, chimeric molecule, GPCR, or cell or tissue expressinga GPCR is attached to a solid phase substrate.

Certain screening methods involve screening for a compound thatmodulates the expression of the GPCRs described herein, or the levels ofnatural ligands, e.g., ASP and stanniocalcins. Such methods generallyinvolve conducting cell-based assays in which test compounds arecontacted with one or more cells expressing the GPCR or ligand and thendetecting an increase or decrease in expression (either transcript ortranslation product). Such assays are typically performed with cellsthat express the endogenous GPCR or ligand.

Expression can be detected in a number of different ways. As describedherein, the expression levels of the protein in a cell can be determinedby probing the mRNA expressed in a cell with a probe that specificallyhybridizes with a transcript (or complementary nucleic acid derivedtherefrom) of the GPCR or protein ligand. Probing can be conducted bylysing the cells and conducting Northern blots or without lysing thecells using in situ-hybridization techniques (see above). Alternatively,protein can be detected using immunological methods in which a celllysate is probed with antibodies that specifically bind to the protein.

Other cell-based assays are reporter assays conducted with cells that donot express the protein. Certain of these assays are conducted with aheterologous nucleic acid construct that includes a promoter that isoperably linked to a reporter gene that encodes a detectable product. Anumber of different reporter genes can be utilized. Some reporters areinherently detectable. An example of such a reporter is greenfluorescent protein that emits fluorescence that can be detected with afluorescence detector. Other reporters generate a detectable product.Often such reporters are enzymes. Exemplary enzyme reporters include,but are not limited to, beta-glucuronidase, CAT (chloramphenicol acetyltransferase), luciferase, beta-galactosidase and alkaline phosphatase.

In these assays, cells harboring the reporter construct are contactedwith a test compound. A test compound that either modulates the activityof the promoter by binding to it or triggers a cascade that produces amolecule that modulates the promoter causes expression of the detectablereporter. Certain other reporter assays are conducted with cells thatharbor a heterologous construct that includes a transcriptional controlelement that activates expression of the GPCR or ligand and a reporteroperably linked thereto. Here, too, an agent that binds to thetranscriptional control element to activate expression of the reporteror that triggers the formation of an agent that binds to thetranscriptional control element to activate reporter expression, can beidentified by the generation of signal associated with reporterexpression.

In one embodiment the invention provides soluble assays using moleculessuch as a domain, e.g., a ligand binding domain, an extracellulardomain, a transmembrane domain (e.g., one comprising seven transmembraneregions and cytosolic loops), the transmembrane domain and a cytoplasmicdomain, an active site, a subunit association region, etc.; a domainthat is covalently linked to a heterologous protein to create a chimericmolecule; a GPCR; or a cell or tissue expressing a GPCR, eithernaturally occurring or recombinant.

In another embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the domain, chimeric molecule,GPCR, or cell or tissue expressing a GPCR is attached to a solid phasesubstrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds arepossible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the signal transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and are appropriate tag binders; see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethylene glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Modulators

Inhibitors and/or activators identified according to the methods of theinvention can be provided from libraries of compounds available from anumber of sources or can be derived by combinatorial chemistryapproaches known in the art. Such libraries include but are not limitedto the available Chemical Director, Maybridge, and natural productcollections. In one embodiment of the invention libraries of compoundswith known or predicted structures can be docked to the human β2ARstructures of the invention. In another embodiment, the libraries forligands binding to the ligand binding site can include carazolol andrelated compounds. In another embodiment, the libraries can include alinker component or moiety. In some embodiments, the linker can includefrom about 10-22 atoms and can include one or more of C, O, N, S, and/orH atoms. In another embodiment, the libraries can include a ligandbinding site (also known as the ligand, agonist, or antagonist bindingpocket) component or moiety. In some embodiments, the libraries caninclude drug-like molecules, i.e., molecules having structuralattributes of one or more compounds known to bind to and/or affect aphysiologic function of a GPCR.

In some embodiments, the invention includes compounds that can be testedas modulators of GPCR activity. Compounds tested as modulators of GPCRscan be any small chemical compound or biological entity. Typically, testcompounds will be small chemical molecules and peptides. Essentially anychemical compound can be used as a potential modulator or ligand in theassays of the invention, although most often compounds can be dissolvedin aqueous or organic (especially DMSO-based) solutions. The assays aredesigned to screen large chemical libraries by automating the assaysteps. The assays are typically run in parallel (e.g., in microtiterformats on microtiter plates in robotic assays). It will be appreciatedthat there are many suppliers of chemical compounds, including Sigma(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis,Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and thelike.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or ligand librariesare then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Russell & Sambrook, all supra),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines,5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

It is noted that modulators that compete with the binding and/oractivity of the known ligands for to human β2AR can be used to treatvarious diseases including, but not limited to, coronary artery disease,atherosclerosis, thrombosis, obesity, diabetes, stroke, and otherdiseases.

In one embodiment, a modulator binds to a site on a GPCR, e.g., a humanβ2AR. In one aspect, the site is a carazolol binding site. In a relatedaspect, the site is a ligand binding site. In another aspect, themodulator has a first moiety that binds to a binding site. In anotheraspect, the first moiety is connected to a linker. In another aspect,the first moiety and the linker are connected to at least one additionalmoiety that binds to a site other than that bound by the first moiety.In another aspect, the two or more moieties are not connected by alinker and are both present in a composition.

Computer-Based Modeling of β2AR

Protein-ligand docking aims to employ principles by which proteinreceptors, e.g., human β2AR, recognize, interact, and associate withmolecular substrates and compounds to predict the structure arising fromthe association between a given compound and a target protein of knownthree-dimensional structure.

In protein-ligand docking, the search algorithm can allow the degrees offreedom of the protein-ligand system to be sampled sufficiently as toinclude the true binding modes. Three general categories of algorithmshave been developed to address this problem of ligand flexibility:systematic methods; random or stochastic methods; and simulationmethods.

Systematic search algorithms attempt to explore all degrees of freedomin a molecule. These algorithms can be further divided into three types:conformational search methods, fragmentation methods, and databasemethods.

In conformational search methods, all rotatable bonds in the ligand aresystematically rotated through 360° using a fixed increment, until allpossible combinations have been generated and evaluated. As the numberof structures generated increases immensely with the number of rotatablebonds (combinatorial explosion), the application of this type of method,in its purest form, is very limited.

Fragmentation methods use two different approaches to incrementally growthe ligands into the active site. One approach is by docking the severalfragments into a site and linking them covalently to recreate theinitial ligand (“the place-and-join approach”). Another approach is bydividing the ligand into a rigid core-fragment that is docked in firstplace and flexible regions that are subsequently and successively added(“the incremental approach”). DOCK (see above) is an example of sdocking programs that use a fragmentation search method.

Database methods using libraries of pre-generated conformations orconformational ensembles to address the combinatorial explosion problem.A example of a docking program using database methods is FLOG whichgenerates a small set of 25 database conformations per molecule based ondistance geometry, that are subsequently subject to a rigid dockingprotocol.

Random search algorithms sample the conformational space by performingrandom changes to a single ligand or a population of ligands. At eachstep, the alteration performed is accepted or rejected based on apredefined probability function. There are three basic types of methodsbased on random algorithms: Monte Carlo methods (MC), Genetic Algorithmmethods (GA), and Tabu Search methods.

Simulation methods employ a rather different approach to the dockingproblem, based on the calculation of the solutions to Newton's equationsof motion. Two major types exist: molecular dynamics (MD) and pureenergy minimization methods.

Scoring functions normally employed in protein-ligand docking aregenerally able to predict binding free energies within 7-10 kJ/mol andcan be divided into three major classes: force field-based, empirical,and knowledge-based scoring functions.

In force-field based scoring, standard force fields quantify the sum oftwo energies: the interaction energy between the receptor and theligand, and the internal energy of the ligand. The energies are normallyaccounted through a combination of a van der Waals with an electrostaticenergy terms. A Lennard-Jones potential is used to describe the van derWaals energy term, whereas the electrostatic term is given by aCoulombic formulation with a distance-dependent dielectric function thatlessens the contribution from charge-charge interactions.

Empirical scoring functions are based on the idea that binding energiescan be approximated by a sum of several individual uncorrelated terms.Experimentally determined binding energies and sometimes a training setof experimentally resolved receptor-ligand complexes are used todetermine the coefficients for the various terms by means of aregression analysis.

Knowledge-based scoring functions focus on following the rules andgeneral principles statistically derived that aim to reproduceexperimentally determined structures, instead of binding energies,trying to implicitly capture binding effects that are difficult to modelexplicitly. Typically, these methods use very simple atomicinteractions-pair potentials, allowing large compound databases to beefficiently screened. These potentials are based on the frequency ofoccurrence of different atom-atom pair contacts and other typicalinteractions in large datasets of protein-ligand complexes of knownstructure. Therefore, their derivation is dependent on the informationavailable in limited sets of structures.

Consensus Scoring combines the information obtained from differentscores to compensate for errors from individual scoring functions,therefore improving the probability of finding the correct solution.Several studies have demonstrated the success of consensus scoringmethods in relation to the use of individual functions schemes.

Using the Protein-ligand docking methods described above, a predictedassociation can be made between a selected chemical library compound(see above for examples) and the binding sites in the human β2ARstructure described in Appendix I (SEQ ID NOS 4-5, 1 and 6-9,respectively in order of appearance). These methods will therefore allowthe generation of a binding profile for any known compound in any of thebinding sites or cavities of the human β2AR based on the simulateddocking of the compound.

In another embodiment, a form of computer-assisted drug design isemployed in which a computer system is used to generate athree-dimensional structure of the candidate class A GPCR based on thestructural information encoded by the amino acid sequence. This willallow use of the methods described above to identify candidate compoundsbased on their ability to dock in one or more of the predicted GPCRstructure binding sites. In one aspect, the input amino acid sequence ofthe GPCR interacts directly and actively with a pre-establishedalgorithm in a computer program to yield secondary, tertiary, andquaternary structural models of the class A GPCR. The models of theclass A GPCR structure are then examined to identify the position andstructure of the binding sites, e.g., a binding pocket. The position andstructure of the predicted binding site(s) is then used to identifyvarious compounds that modulate ligand-receptor binding using themethods described above.

The three-dimensional structural model of the GPCR is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a GPCR polypeptide intothe computer system. The amino acid sequence represents the primarysequence or subsequence of the protein, which encodes the structuralinformation of the protein. At least 10 residues of the amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the GPCR is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art. Any method ofprotein structure modeling such as ab-initio modeling, threading orsequence-sequence based methods of fold recognition. In one embodiment,the AS2TS system of protein structure modeling is used. In otherembodiments, a sequence alignment in combination with a thresholdprotein sequence similarity to determine a set of protein sequences forwhich to model protein structure is used. In one aspect, sequencealignments are generated for the set of sequences to be modeled withsequences of proteins with solved empirical structure in a proteinstructure databank known to one of skill in the art. If the sequences tobe modeled have a sufficient similarity to one or more sequences withknown protein structure, then the three dimensional structure of thesequence can be modeled.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the GPCR of interest. In one embodiment, software can lookat certain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as “energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

In another embodiment, protein structure alignments can be used todetermine the structure of GPCRs using the known structure of the β2AR(Appendix I) (SEQ ID NOS 4-5, 1 and 6-9, respectively in order ofappearance). Protein structure alignments preferably are sets ofcorrespondences between spatial co-ordinates of sets of carbon alphaatoms which form the ‘backbone’ of the three-dimensional structure ofpolypeptides, although alignments of other backbone or side chain atomsalso can be envisioned. These correspondences are generated bycomputationally aligning or superimposing two sets of atoms order tominimize distance between the two sets of carbon alpha atoms. The rootmean square deviation (RMSD) of all the corresponding carbon alpha atomsin the backbone is commonly used as a quantitative measure of thequality of alignment. Another quantitative measure of alignment is thenumber of equivalent or structurally aligned residues.

In another embodiment, a GPCR structure is calculated based on thesolved structure of the human β2AR by computationally aligning orsuperimposing two sets of atoms to minimize distance between the twosets of carbon alpha atoms (i.e., the alpha carbon atoms of the humanβ2AR and an unknown GPCR structure), followed by one or more ofsimulated annealing and energy minimization. The result of thiscalculation is a computed structure for a GPCR that provides atomicco-ordinates for the alpha carbon backbone as well as side chain atoms.

A variety of methods for generating an optimal set of correspondencescan be used in the present invention. Some methods use the calculationof distance matrices to generate an optimal alignment. Other methodsmaximize the number of equivalent residues while RMSD is kept close to aconstant value.

In the calculation of correspondences, various cutoff values can bespecified to increase or decrease the stringency of the alignment. Thesecutoffs can be specified using distance in Angstroms. Depending on thelevel of stringency employed in the present invention, the distancecutoff used is less than 10 Angstroms or less than 5 Angstroms, or lessthan 4 Angstroms, or less than 3 Angstroms. One of ordinary skill willrecognize that the utility of stringency criterion depends on theresolution of the structure determination.

In another embodiment of the present invention, the set ofresidue-residue correspondences are created using a local-globalalignment (LGA), as described in US Patent Publication Number2004/0185486. In this method, a set of local superpositions are createdin order to detect regions which are most similar. The LGA scoringfunction has two components, LCS (longest continuous segments) and GDT(global distance test), established for the detection of regions oflocal and global structure similarities between proteins. In comparingtwo protein structures, the LCS procedure is able to localize andsuperimpose the longest segments of residues that can fit under aselected RMSD cutoff. The GDT algorithm is designed to complementevaluations made with LCS searching for the largest (not necessarycontinuous) set of ‘equivalent’ residues that deviate by no more than aspecified distance cutoff.

Using the protein structure alignments described above, the structure ofhuman β2AR in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively inorder of appearance) can be used as a model on which to discern thestructure of other GPCRs and/or their predicted ligand-binding sites.

Once the GPCR structure has been generated, a binding pocket can beidentified by the computer system. Computational models seek to identifythe regions by characterization of the three dimensional structure ofthe GPCR. Some methods of identifying a binding pocket use triangulationsuch as weighted Delaunay triangulation to determine pocket volumes(castP). Other methods use spheres to determining protein pocket volumes(Q-site-finder, UniquePocket). Conserved binding-site identificationseeks to identify conserved regions such as a binding pocket throughassociating the residues which form the aforementioned regions withconserved residues in homologous protein sequences or structures, e.g.,through the use of sequence alignments.

One method of identifying a binding pocket in a GPCR entails filling thethree dimensional protein structures with spheres, creating a “negativeimage” of the structure. A cutoff distance, such as 8 Angstroms, is usedto determine spheres which interact with residues. Spheres are labeledas conserved or not-conserved based on their interaction with residueswhich form a conserved binding site. The conserved spheres are clusteredbased on their three dimensional co-ordinates to identify a set ofspheres with interact with conserved residues and are proximal in threedimensional space forming a cluster. Three-dimensional structures forpotential compounds are generated by entering chemical formulas ofcompounds. The three-dimensional structure of the potential compound isthen compared to that of the GPCR protein ligand-binding site(s) (e.g.,a binding pocket) to identify compounds that bind to the GPCR bindingsite(s). Binding affinity between the GPCR binding site(s) and thecompound is determined using energy terms to determine which ligandshave an enhanced probability of binding to the protein.

It should be noted that the language used in the specification has beenprincipally selected for readability and instructional purposes, and cannot have been selected to delineate or circumscribe the inventivesubject matter. Accordingly, the disclosure of the present invention isintended to be illustrative, but not limiting, of the scope of theinvention, which is set forth in the claims.

Reference will now be made in detail to particularly preferredembodiments of the invention. Examples of the preferred embodiments areillustrated in the following Examples section.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols. A and B (1992).

Example 1 Crystallization of a β2AR Protein Using a Lipidic CubicPhase/Sterol Method

This Example describes the generation of diffraction-quality crystals ofa β2AR protein, specifically crystals of β2AR-T4L, a fusion protein ofhuman β2AR with T4 lysozyme, bound to carazolol. A detailed descriptionof the protein and its synthesis is provided in Example 4. Briefly, T4lysozyme was generated by three distinct modifications to β2AR: (1) afusion protein was created by replacement of the third intracellularloop with T4L, (2) the carboxyl terminal 48 amino acids were deleted,and (3) a glycosylation site at Asn187 was eliminated through aglutamate substitution. β2AR-T4L was expressed in Sf9 insect cells,solubilized in 1% dodecylmaltoside, and purified by sequential antibodyand ligand affinity chromatography.

LCP Crystallization Setup

Protein solution (30 mg/ml) was mixed with a host lipid or lipid mixturetypically in 2:3 ratio by volume ratio using a syringe mixer (Cheng, etal., Chem Phys Lipids 95, 11 (1998). Upon mixing (˜100 passages, 2-3min) the sample spontaneously formed homogeneous transparent cubicphase, which was directly used in crystallization trials. Robotic trialswere performed using an in meso crystallization robot (Cherezov, et al.,Acta Cryst D 60, 1795 (2004)). Six-well glass sandwich plates (Cherezov,et al., Acta Cryst D 60, 1795 (2004); Cherezov, J Appl Cryst 36, 1372(2003)), were filled with 25 or 50 nL protein-laden lipidic cubic phasedrops overlaid by 800 μL, of precipitant solution in each well andsealed with a glass coverslip. Manual setups were performed in Impactmicrobatch plates (Hampton Research cat#HR3-293), Innovaplate SD-2sitting drop plates (Hampton Research cat#HR3-083) or VDX48 hanging dropplates (Hampton Research cat#HR3-275). Modified repetitive syringedispenser (Cherezov, et al., J Appl Cryst 38, 398 (2005)), coupled witha gas-tight 10 μL syringe was used to deliver 70 nL of cubic phase dropsper well to which 1-2 μL of precipitant solution was added with amicrovolume pipette. Reservoirs of the Innovaplate and VDX48 plates werefiled with 50 and 100 μL of precipitant respectively. All operationsstarting from mixing lipid and protein were performed at roomtemperature (˜21-23° C.). After setup, plates were transferred into anautomatic incubator/imager (Rocklmager 1000, Formulatrix Inc.)maintained at 20° C. Plates were imaged every 12 hours for the first 3days, then every day until 7 days and after that on the 10th and on the14th day.

Initial Hits

Initial trials were performed using protein solution at a concentrationof 30 mg/mL mixed with monoolein as a host lipid against a set of 6commercial screens (Index HT, SaltRx HT and MemFac HT from HamptonResearch, JCSG+ and MbClass from Nextal, and MemSys&MemStart fromMolecular Dimensions) set up in duplicates. Initial hits detected inthree different wells contained extremely small, <5 μm, needle-likebirefringent crystal showers. The detection of such small colorlessprotein crystals in LCP was made feasible by specially developed glasssandwich plates with optimized optical properties (Cherezov, et al.,Acta Cryst D 60, 1795 (2004); Cherezov et al., J Appl Cryst 36, 1372(2003)). Hit conditions were similar by chemical composition containing30% v/v PEG 400 as a precipitant, low concentration of Li sulfate and abuffer at pH 6.5 or 7.5.

Optimization

Crystal condition optimization is well known to practioners in the artof x-ray crystallography. What follows is a specific example of agenerally applicable optimization approach in which one or more of thecrystallization mixture components is systematically varied inconcentration or substituted by a chemical analog. Initial rounds ofoptimization were focused on varying concentration of the mainprecipitant, PEG 400, buffer pH and identity, and salt identity andconcentration. As a result, Li sulfate was replaced with Na sulfate anduseful concentration and pH ranges were established. Crystals were stillrather small reaching ˜15×5×1 μm in size.

Further, lipid and soluble additives were searched for and optimizedsimultaneously. Three different host lipids (monopalmitolein,monovaccenin and monoolein), five lipid additives to monoolein host(DOPE, DOPE-Me, DOPC, Asolectin and cholesterol) and 96-well solubleadditives screen were tried in different combinations along with thepreviously found basic crystallization conditions. One of the solubleadditives, 1,4-butanediol, stood out, but only when it was used incombination with lipid additives, DOPE or cholesterol. When DOPE wasused as an additive the crystals grew as thin plates (40×7×2 μm), whilewhen cholesterol was used the crystals grew as small rods (30×5×5 μm).DOPE was dropped out in favor of cholesterol in the subsequentoptimization steps.

Final optimization required fine tuning concentrations of all components(protein, PEG 400, Na sulfate, 1,4-butanediol, cholesterol, buffer pH).At the final stages of optimization, higher concentrations of protein,e.g., 50 mg/mL protein solution, were preferred. Decreasing the volumeof lipidic cubic phase per trial from 50 to 20 nL consistently producedlarger crystals. The best crystals (40×20×5 μm; FIG. 1) were obtained in30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol inmonoolein as the host lipid. Thus, in another aspect, the inventionprovides a method of directly adding a lipid additive (e.g.,cholesterol, DOPE) to the host lipid prior to combining with the proteinmixture, thereby significantly improving the size and quality of LCPgrown crystals. Previously, exogenous lipid had been added only to theprotein solution prior to combining the protein solution with the hostlipid (Luecke, et al., Science 293, 1499 (2001)).

To limit the range of precipitant and additives concentrations used forscreening, previously published phased diagrams were used, e.g., formonoolein (Qiu, et al., Biomaterials 21, 223 (2000)), and monovaccenin(Qiu, et al., J. Phys. Chem. B 102, 4819 (1998)), and the effects onmonoolein phase behavior of soluble (Cherezov, et al., Biophys J 81, 225(2001)), and lipid additives (Cherezov, et al., Biophys J 83, 3393(2002)). Certain lipid mixtures required pre-screening their phasebehavior by microscope visualization and by X-ray, prior to being mixedwith the protein for crystallization trials. Thirty-three 96-welloptimization screens were used in combinations with different lipidmixtures, resulting in over 15,000 trials. This throughput was madefeasible through the use of an in meso crystallization robot andautomatic imager.

Typically, the best crystals grown under lipidic cubic phase conditionsappear at the boundary between the cubic and the sponge phase (Cherezov,et al., J Mol Biol 357, 1605 (2006); Wadsten, et al., J Mol Biol 364, 44(2006)). When crystals are close to the phase boundary, equally goodcrystals are obtained in either phase.

Crystal Harvesting

Crystals were harvested directly from the glass sandwich plates (FIG. 2)because this method provided results superior to those obtained withmicrobatch or vapor diffusion plates. These plates have beenspecifically designed to perform optimally only at the screening andoptimization stages (Cherezov, et al., Acta Cryst D 60, 1795 (2004);Cherezov and M. Caffrey, J Appl Cryst 36, 1372 (2003)). No harvestinghas been previously attempted from them, due to the difficulties inseparating glass slides strongly bound by a high performance doublesticky tape. Thus, in another aspect, the invention disclosed hereinprovides a special technique for opening individual wells and harvestingcrystals from them. A corner of a capillary cutting stone (in thisinstance, from Hampton Research) was used to scratch the top glassaround the perimeter of the well. Gently pressing the glass slide justoutside the scratch allows propagation of the scratch through the depthof the glass. The glass slide was then broken in one point just outsideof the scratched perimeter using a sharp needle. This hole was used tolift up the glass slide and expose the cubic phase for harvesting. Anextra drop of ˜5 μL of precipitant was added to the well to reducedehydration. Using this technique, it was possible to open up andharvest crystals successfully from more than 80% of attempted wells.

Crystals were scooped directly from the lipidic cubic phase using 30 μmaperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care wastaken to drag as little as possible lipid around the crystal to decreasethe unwanted background. Attempts to dissolve the lipids, either byincreasing concentration of PEG400 or using a mineral oil, typicallyresulted in decreasing the diffraction power of the crystals.

Data Collection

During screening for diffraction of these crystals at APS beamline GM/CACAT (FIG. 3), the crystals themselves could not be observed in themounted loops (FIG. 1 a). Therefore, a systematic screening of the loopmaterial with varying beams was conducted to identify the crystal in theloop. Optimization of the diffraction with a low x-ray dose was thenused to center the crystals and eventually allow for complete datacollection using the 10 μm×10 μm minibeam setup at GM/CA CAT. Thecomplete data set is then compared to data filtered by a sigma cutoff(see Table 1). All of the data was used in structure solution andrefinement.

TABLE 1 # Observed # Unique Resolution refl refl Redundancy CompletenessR_(sym) I/SIGMA R-meas Rmrgd-F Signal/noise −3 10   3352 333 10.1 87.40%6.60% 23.25 6.90% 2.70% 8   3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80%6   10480 1003 10.4 99.60% 9.90% 20.01 10.50% 4.00% 3   126008 1196810.5 99.80% 13.30% 14.01 14.00% 6.60% 2.8 33158 3130 10.6 100.10% 38.00%6.42 39.90% 18.70% 2.7 19702 1893 10.4 99.70% 49.50% 4.96 52.10% 24.90%2.6 23772 2275 10.4 99.90% 60.20% 4.07 63.40% 30.10% 2.5 14108 2558 5.599.30% 58.90% 2.69 65.10% 51.80% 2.4 14672 3060 4.8 99.10% 67.80% 2.1875.70% 62.80% total 248843 26574 9.4 99.50% 12.70% 9.62 13.40% 11.40%Signal/noise 0 10   3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8  3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6   10480 1003 10.4 99.60%9.90% 20.01 10.50% 4.00% 3   125628 11923 10.5 99.40% 13.30% 14.0614.00% 6.50% 2.8 32679 3077 10.6 98.40% 37.60% 6.54 39.50% 17.70% 2.719346 1849 10.5 97.40% 48.80% 5.08 51.30% 23.40% 2.6 23201 2210 10.597.00% 58.90% 4.2 62.00% 28.00% 2.5 13461 2406 5.6 93.40% 56.40% 2.8862.30% 45.90% 2.4 13833 2827 4.9 91.50% 64.10% 2.4 71.50% 54.30% total245571 25982 9.5 97.30% 12.60% 9.85 13.30% 10.80%

Example 2 Using the LCP/Sterol Method to Generate Additional MembraneProtein Crystals

In addition to the β₂AR-T4L/carazolol structure (Examples 1, 3, and 4),the LCP/sterol matrix has successfully been used to crystallize adiversity of receptor-ligand systems.

1. β₂AR-T4L^((E122W))

A thermally-stabilized construct of β₂AR-T4L comprising an E122Wmutation has been crystallized in the presence of both agonist andantagonist ligands including: alprenolol, timolol, clenbuterol andcarazolol. For lipidic cubic phase (LCP) crystallization ofNAR-T4L^((E122W)), robotic trials were performed using an in mesocrystallization robot (Cherezov et al., 2004). Glass sandwich plates in96-well format (Cherezov and Caffrey, 2003; Cherezov et al., 2004) werefilled with 25 or 50 nL protein-laden LCP drops overlaid by 0.8 μL ofprecipitant solution in each well and sealed with a glass coverslip. Alloperations starting from mixing lipid and protein were performed at roomtemperature (˜21-23° C.). Crystals were obtained in 28% (v/v) PEG 400,0.3 M potassium formate, 0.1 M Bis-tris propane pH 7.0 and saturatingconcentrations of ligand (e.g., 2 mM in the case of timolol) using 10%(w/w) cholesterol in monoolein as the host lipid. Diffraction data werecollected on all four ligand complexes (see FIG. 4), and structures weredetermined for alprenolol (3.2 Å), timolol (2.8 Å), and carazolol (2.8Å).

2. A_(2A)R-T4L

The applicability of the monoolein cholesterol system in thecrystallization of non-biogenic amine receptors has also beendemonstrated with the structural determination of the human A_(2A)adenosine receptor (A_(2A)R-T4L) bound to a high affinity selectiveantagonist, ZM241385, to 2.6 Å resolution. See FIG. 4. For lipidic cubicphase (LCP) crystallization of the human A2A adenosine receptor in meso,glass sandwich plates (Cherezov, et al., Acta Crystallogr D BiolCrystallogr, 60, 1795 (2004)) were filled with 50 nlreceptor-cholesterol-monoolein LCP drops overlaid by 0.8 μl ofprecipitant solution in each well and sealed with a glass coverslip.Lipid:receptor LCP mixture typically contained monoolein:cholesterol(54%:6% (w/w)) and receptor (40% (w/w)). Crystallization set-ups wereperformed at ambient temperature (22±2° C.). Plates were incubated andimaged at 20° C. using an automated incubator/imager (RockImager 1000,Formulatrix). Data-collection quality crystals 100 μm×10 μm×5 μm) wereobtained in 30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate(range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5to 6.5) and 200 μM ZM241385. The protein crystallized in the primitivemonoclinic space group P2₁ with one molecule per asymmetric unit and anestimated solvent content of 52%.

3. β₂AR^((E122W))

Initial crystals of β₂AR^((E122W)), lacking inserted T4 lysozyme, havealso been obtained. The protein was extracted from insect cell membranesusing a mixture of 0.5% w/v dodecyl maltoside (DDM), 0.1% w/vcholesteryl hemisuccinate (CHS) and 1 mM timolol. Timolol was maintainedat 1 mM throughout the first steps of the purification. The extractedprotein was purified by binding overnight to Talon™ immobilized metalaffinity resin followed by a standard washing and elution with 200 mMimidazole. Adenosine triphosphate at 5 mM in combination with 10 mMMgCl₂ was used to eliminate chaperone protein contamination. Elutedprotein was concentrated to 2.5 mL and desalted into a 0 mM imidazolebuffer using a PD10 desalting column (GE-Biosciences). Protein was thenbound to 100 μL of Ni-sepharose immobilized metal affinity resin in thepresence of PNGase (New England Biolabs) to remove glycosylation, andincubated overnight. After incubation the protein was washed on thecolumn and timolol was replaced by carazolol for structure solution. Theprotein bound to carazolol was eluted from the Ni-Sepharose column,treated with 100 mM Nacitrate pH 7.5, and concentrated to 50 mg/mL.

The protein solution was then mixed with monoolein containing 10%cholesterol at a ration of 40:60% w/w protein to lipid to generate thelipidic cubic phase used in crystallization trials. The LCP lipidcontaining protein was dispensed onto glass sandwich crystallizationplates at a volume of 20 nL to which 1 μL of crystallization solutionwas added. The entire experiment in 96 well format was then covered byan additional glass plate which was fastened to the first by virtue ofdouble back sticky tape. Initial crystals have been obtained after 24hours by addition of a solution containing 35% v/v PEG 400, 100 mM NaSO4100 mM Bis tris propane pH 7 and 8% 2,6 hexanediol.

By way of a prophetic example, optimized crystals of β₂AR^((E122W))obtained by this method are screened for their ability to diffract athigh resolution, e.g., less than 3.5 Å or, more preferably, less than 3Å. Guidance for optimization is provided by the optimization protocolsset forth herein and in the examples. In combination with the teachingprovided herein, one skilled in the art will readily identifyappropriate beam settings to obtain diffraction patterns from which adetailed molecular structure of the optimally crystallized protein canbe solved.

4. CXCR4-T4L

CXCR4, also called fusin, is a GPCR protein specific forstromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule endowedwith potent chemotactic activity for lymphocytes. This Example teachesprophetically how the methods of the invention may be used to generatediffraction quality crystals of a fusion protein comprising CXCR4(CXCR4-T4L).

The cDNA encoding CXCR4 is synthesized by outsourcing to DNA2.0 wherethe DNA was optimized for human codon usage, elimination of transcribedRNA secondary structure, elimination of ribosome binding sites andavoidance of common restrictions sites used in subsequent cloningprocedures. Two initial variants are contracted to be synthesized, thefirst encoding a wild-type full-length receptor and the second a fulllength receptor with a fusion protein located between transmembranehelix V and helix VI, effectively eliminating the third intracellularloop (3IL) region of the receptor. In the case of CXCR4, T4-lysozyme(T4L) is the fusion protein fused in the place of the 3IL. A set ofguidelines is followed for the incorporation of T4L into the fusionprotein which minimizes the possibility of structural disruption andconcomitant effects on protein expression and stability. Briefly, the 5′insertion point for the fusion protein takes precedence over the 3′insertion point and is located 66 nucleotides (22 residues) downstreamof the codon for a conserved proline on helix V of the receptor. If the3IL section of the receptor is large, the 3′ fusion point is set 87nucleotides (29 residues) upstream from the codon for the familyconserved proline on helix VI. However, as is the case for CXCR4, wherethe 3IL loop is small, cDNA for the fusion protein is inserted directlyinto the 3IL loop position dictated by spacing from helix V with noresulting excision of intervening nucleotides. Specifically, T4L isinserted into CXCR4 based on spacing between a conserved proline onhelix V and a C-terminal truncation was generated based on literatureprecedence. Each synthesized cDNA is flanked by an out of frame AscI(GGCGCGCCG) restriction site on the 5′ end and an in frame FseI(GGCCGGCC) on the 3′ end for sub-cloning into a set of four expressionvectors. Viral DNA is then generated, amplified from these vectorsaccording to standard protocols and titered using flow cytometry tomeasure the population of cells expressing the virally encoded GP64protein.

Protein Expression of CXCR4-T4L

With titered virus in hand, small scale expression trials are carriedout in a volume of 5 mL/experiment. Expression levels are assessed usingflow cytometry to measure the mean fluorescence intensity (MFI) andpercentage of cells expressing the FLAG epitope encoded by theexpression screening vectors. Expressing cells are tested with andwithout permeabilization to generate a ratio between protein inserted inthe plasma membrane and protein inside the cellular traffickingmachinery. A correlation between cell surface expression and overallprotein expression is demonstrated, as well as a correlation betweenstability and the ratio of cell surface expression/total expression. Inaddition to these assays, small scale purification after solubilizationwith dodecyl maltoside (DDM) is carried out to determine the quantity ofrecoverable protein as well as the quality as measured by size exclusionchromatography. Based on these data it was apparent that the T4L fusedreceptor is expressing and that it is dimerizing in a ligand independentmanner, an indicator of C-terminal non-specific interactions in otherreceptors. Thus, a C-terminal truncation mutant of CXCR4 is generated.

Protein Purification

The C-terminal truncation of T4L fused CXCR4 was scaled up to productionsized expression (5-10L of cell culture) and further processed by largescale purification efforts intended for crystallization trials. Briefly5-10 L of cells culture are centrifuged and washed with PBS followed byfreezing at −80° C. The frozen cellular material is then resuspended in820 mL of lysis buffer (10 mM Hepes pH 7.5, 10 mM MgCl₂, 20 mM KCl)supplemented with protease inhibitor (Roche). The cell suspension islysed by 20 strokes of a dounce homogenizer and centrifuged at 45,000rpm in Ti45 ultracentrifuge for 30 minutes. The resulted pellet wasseparated from the supernatant, resuspended and the process repeated sixtimes to ensure complete removal of soluble protein material. On thefinal resuspension step the membranes were resuspended in 100 mL oflysis buffer containing 40% v/v glycerol, homogenized with 20 strokes ofa dounce homogenizer and frozen in 10 mL aliquots at −80° C. forindefinite storage.

For solubilization and purification, each 10 mL aliquot of frozenmembranes is resuspended to 25 mL using lysis buffer to which 100 uMAMD070 ligand is added in addition to protease inhibitor at 2× workingconcentration and 2 mg/mL iodoacetamide. The membranes are allowed tothaw and incubate with ligand at an appropriate temperature for at least30 minutes. After the incubation the mixture is diluted two-fold with a2× solubilization buffer containing 100 mM Hepes pH 7.5, 1M NaCl, 2% w/vDDM 0.2% w/v CHS. The solubilization is allowed to proceed withagitation for at least 2 hours at 4° C. after which insoluble materialis separated by centrifugation and discarded. The supernatant isisolated and allowed to bind to 0.5 mL of Talon (Clontech) IMAC resincharged with Co²⁺ in the presence of 20 mM imidizole buffered to 7.5 and800 mM NaCl. Binding to the Talon IMAC resin is allowed to proceed withagitation at least 4 hours but most commonly overnight. After binding,the slurry is poured into a gravity column and the resin is separatedfrom the supernatant. The resin is then washed with 80 column volumes(CV) of wash buffer (50 mM Hepes pH 7.5, 800 mM NaCl, 20 mM Imidizole,0.1% w/v DDM, 0.01% w/v CHS and 100 uM AMD070 (or receptor appropriateligand). After the initial wash the resin is further treated to adjustthe NaCl concentration to 500 mM and to increase the ligandconcentration to 300 uM. The protein is then eluted from the resin using200 mM Imidazole and concentrated to 2.5 mL for removal of the excessimidizole with a PD10 desalting column (GE Biosciences). The ligandconcentration is increased to 500 uM and the protein is bound to 100 uLof Ni-Sepharose IMAC resin in the presence of 20,000 units of PNGase(NEB) an endoglycosidase capable of removing N-linked glycosylation. Theprotein is allowed to bind to the resin and deglycosylate for 6 hoursafter which the resin is washed with imidizole free elution buffer (50mM Hepes pH 7.5, 500 mM NaCl, 0.05% w/v DDM, 0.01% w/v CHS and 1 mM AMD070). After the washing step the protein was eluted from the resin usingthe same buffer but including 200 mM imidizole. After elution theprotein is normally concentrated to approximately 50 mg/mL and testedfor integrity by SEC. Crystallizable protein should be >90% free ofheterogeneity as judged by SDS-PAGE and contain no detectable aggregatedspecies at high protein concentrations as judged by SEC. If the proteinremains of high quality it is reconstituted into lipidic cubic phasecontaining cholesterol. The reconstituted protein is then dispensed ontoglass sandwich crystallization plates and tested for crystallizationusing the screening methodology described in this Example and Example 1.After mixing, the protein-laden lipidid cubic phase mixture willcomprise 3.6-7.2% w/w sterol, 56.5-52.8 w/w % Monoolein and 40% w/wprotein solution (a 3:2 ratio of lipid mixture to protein). Initialcrystallization conditions use PEG 400 between 25-35%, a salt between50-500 mM, and a pH between 5.0-7.5.

Example 3 High Resolution Crystal Structure of a Human β₂-Adrenergic GProtein-Coupled Receptor T4 Lysozyme Fusion Protein

The engineering, functional properties, expression and purification ofcrystallization grade β2AR-T4L protein are described in more detail inExample 4. Briefly, β2AR-T4L was generated by three distinctmodifications to β2AR: (1) a fusion protein was created by replacementof the third intracellular loop with T4L, (2) the carboxyl terminal 48amino acids were deleted, and (3) a glycosylation site at Asn187 waseliminated through a glutamate substitution. β2AR-T4L was expressed inSf9 insect cells, solubilized in 1% dodecylmaltoside, and purified bysequential antibody and ligand affinity chromatography. Using themodified lipidic cubic phase (LCP) crystallization procedure describedherein, wherein crystals are grown from a cholesterol-doped monooleincubic phase, β2AR-T4L crystals were obtained that diffract to aresolution of 2.2 Å. The structure was solved at 2.4 Å resolution.Compared to crystallization in detergents, LCP provides a more native,lipid environment for crystallization, as well as a confinement ofprotein molecules to two-dimensional membrane sheets that may facilitatethe crystallization process through the formation of Type I packinginteractions (Caffrey, Curr Opin Struct Biol 10, 486 (2000);Deisenhofer, EMBO J 8, 2149 (1989); Landau et al., Proc Natl Acad Sci US A 93, 14532 (1996)).

Methods

Lipidic Cubic Phase Crystallization.

Crystals of engineered human β2AR 032AR-T4L) grown from bicelles couldnot be optimized beyond 3.5 Å resolution (FIG. 5). Lipidic cubic phase(LCP) crystallization trials were therefore performed using an in mesocrystallization robot (Cherezov, et al., Acta Crystallogr D BiolCrystallogr 60, 1795 (2004)). 96-well glass sandwich plates (Cherezov,et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov,et al, J Membr Biol 195, 165 (2003)) were filled with 25 or 50 nLprotein-laden LCP drops overlaid by 0.8 μL of precipitant solution ineach well and sealed with a glass coverslip. All operations startingfrom mixing lipid and protein were performed at room temperature(˜21-23° C.). Crystals were obtained in 30-35% (v/v) PEG 400, 0.1-0.2 Msodium sulfate, 0.1 M Bis-tris propane pH 6.5-7.0 and 5-7% (v/v)1,4-butanediol using 8-10% (w/w) cholesterol in monoolein as the hostlipid (FIG. 6A). Addition of cholesterol and 1,4-butanediol dramaticallyimproved crystals size and shape, thereby enabling high-resolutiondiffraction. In this instance, additions of phospholipids(dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine,asolectin) to the main host LCP lipid monoolein (either alone or incombination with cholesterol) failed to improve crystal quality.

Crystal Harvesting

The average size of the harvested crystals was 30×15×5 μm (largestcrystal was 40×20×7 μm). Crystals were harvested directly from the glasssandwich plates, even though these plates have been specificallydesigned for screening and optimization (Cherezov, et al., ActaCrystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J MembrBiol 195, 165 (2003)). Crystals were scooped directly from the LCP using30 or 50 μm aperture MiTeGen MicroMounts and plunged into liquidnitrogen. Care was taken to drag as little as possible lipid around thecrystal to decrease unwanted background scattering. Attempts to dissolvethe lipids, either by increasing concentration of PEG 400 or using amineral oil, typically resulted in a decrease in diffraction power ofthe crystals.

Data Collection

X-ray data were collected on the 231D-B beamline (GM/CA CAT) at theAdvanced Photon Source, Argonne, IL using a 10 μm minibeam (wavelength1.0332 Å) and a MarMosaic 300 CCD detector (FIG. 6B). Several completedatasets were collected from single crystals at resolution between 2.8and 3.5 Å using 5× attenuated beam, 5 s exposure and 1° oscillation perframe. However, some crystals diffracted to a maximum of 2.2 Åresolution upon 5 s exposure with 1× attenuated beam. Therefore, 10-20°wedges of high-resolution data were collected from more than 40 crystals(some of the crystals were large enough to allow 2-3 translations). 31of the best datasets from 27 independent crystals were then combined andscaled against the lower resolution full dataset to obtain complete 2.4Å data.

One of the challenges during data collection was visualization ofcolorless microcrystals within an opaque frozen lipid phase and aligningthem with the 10 μm minibeam. Because the crystals could not beadequately visualized through the inline optics at the beamline,alignment-by-diffraction techniques were employed. The present inventionprovides, in one aspect, an optimized crystal search algorithm to locatethe crystals without the minibeam. First, the area of the loopcontaining lipid was scanned in the vertical direction with a highlyattenuated and slitted 100×25 μm beam. When diffraction was found, thecrystal location was further confined by two additional exposures to anarea of ˜50×25 μm. This area was further coarse-scanned with thecollimated and 10× attenuated minibeam using 15 μm steps, following byfine-tuning the position using 5 and 2 μm steps. After locating thecrystal in one orientation the loop was rotated 90° and the procedurewas repeated. Typically during alignment the crystal was exposed ˜10times using 10× attenuated beam and 2 s exposures.

Data Processing

A 90% complete, 2-fold redundant monoclinic dataset was processed fromone crystal diffracting to 2.8 Å resolution. Initial indexing of latticeparameters in spacegroup C2 and crystal orientation were performed usingHKL2000 (Otwinowski, et al, in Methods in Enzymology C. W. J. Carter, R.M. Sweet, Eds. (Academic Press, New York, 1997), vol. 276, pp. 307-326).The refined lattice parameters and space group were implemented in thedata processing program XDS for spot integration which models errorexplicitly for radiation decay, absorption, and rotation (Kabsch, J ApplCrystallogr 26, 795 (1993)). Because data was collected using a 10 μmbeam from microcrystals, maintaining the crystal orientation at the beamcenter during data collection was especially problematic. It appearedthat XDS modeled the crystal orientation error upon rotation about thephi axis better than other data processing programs that were tried,resulting in better merging statistics. In addition to rotational error,the radiation decay was also an issue that was partially corrected bythe XDS processing program, enabling a more reliable scaling of datasetsfrom different crystals and translations of crystals. The 2.8 Å data wasused as a scaling reference for incorporation of additional wedges ofdata collected at a much higher exposure. Each new dataset was indexedin XDS using the original unit cell parameters as constants which werethen refined along with the crystal orientation, beam geometry, andmosaicity parameters. The refinement was generally stable, resulting invery similar unit cell constants which enabled subsequent scaling. Allof the integrated wedges of data were then tested individually againstthe scaling reference set and included in the final scaled dataset ifthe merging statistics remained acceptable upon incorporation of thedata. In total, 31 wedges of data from 27 crystals were combined withthe scaling reference dataset, 22 of which diffracted to a resolution of2.4 Å or better. Each of the higher resolution datasets were exposed toa much larger dose of radiation resulting in a rapid decay in intensity.Typically 10°-20° wedges were collected from each crystal ortranslation, 5°-7° of which had diffraction data to 2.4 Å. The finalmerging statistics for the dataset are shown in Table 2. Based on themean F/σ(F) of reflections near the three crystallographic axes, theeffective resolution is estimated to be 2.4 Å along b* and c* and 2.7 Åalong a*. The anisotropy results in the high merging R factors in thelast few resolution shells despite the significant I/σ(I) values. Theanisotropy is either an inherent property of the crystals or the resultof a preferential orientation of the crystals within the mounting loop.Thus, the higher resolution shells were filled in anisotropically byincorporation of the additional data at high exposure levels, while thelower resolution shells have a very high redundancy and low anisotropy.

TABLE 2 Data processing statistics from XDS. A comparison is madebetween data filtered by a sigma cutoff and the complete set. All of thedata was used in structure solution and refinement. # Observed # UniqueI/ Resolution refl refl Redundancy Completeness R_(sym) SIGMA R-measRmrgd-F Signal/noise ≧ −3 10   3352 333 10.1 87.40% 6.60% 23.25 6.90%2.70% 8   3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6   10480 100310.4 99.60% 9.90% 20.01 10.50% 4.00% 3   126008 11968 10.5 99.80% 13.30%14.01 14.00% 6.60% 2.8 33158 3130 10.6 100.10% 38.00% 6.42 39.90% 18.70%2.7 19702 1893 10.4 99.70% 49.50% 4.96 52.10% 24.90% 2.6 23772 2275 10.499.90% 60.20% 4.07 63.40% 30.10% 2.5 14108 2558 5.5 99.30% 58.90% 2.6965.10% 51.80% 2.4 14672 3060 4.8 99.10% 67.80% 2.18 75.70% 62.80% Total248843 26574 9.4 99.50% 12.70% 9.62 13.40% 11.40% Signal/noise ≧ 0 10  3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8   3591 354 10.1 99.40%7.60% 22.6 8.00% 2.80% 6   10480 1003 10.4 99.60% 9.90% 20.01 10.50%4.00% 3   125628 11923 10.5 99.40% 13.30% 14.06 14.00% 6.50% 2.8 326793077 10.6 98.40% 37.60% 6.54 39.50% 17.70% 2.7 19346 1849 10.5 97.40%48.80% 5.08 51.30% 23.40% 2.6 23201 2210 10.5 97.00% 58.90% 4.2 62.00%28.00% 2.5 13461 2406 5.6 93.40% 56.40% 2.88 62.30% 45.90% 2.4 138332827 4.9 91.50% 64.10% 2.4 71.50% 54.30% Total 245571 25982 9.5 97.30%12.60% 9.85 13.30% 10.80% R_(sym) = Σ_(hkl)|I(hkl) −<I(hkl)>|/Σ_(hkl)(hkl), where <I(hkl)> is the mean of the symmetryequivalent reflections of I(hkl); R-meas = redundancy independent Rsym;Rmrgd-F = indication of amplitude quality. See, e.g., Murshudov, et al,Acta Crystallogr D D53, 240 (1997).

Structure Solution and Refinement

Initial phases for β2AR-T4L were obtained by molecular replacement usingboth T4-lysozyme (PDB ID Code 2LZM) and a polyalanine model of therhodopsin seven-transmembrane bundle (PDB ID Code 1U19) as searchmodels. It was necessary to trim the lysozyme search model to removeresidues 12-71 as that domain had shifted conformations relative to thelarger section. This domain was later reintroduced to the model byfitting into observed density. Molecular replacement was carried outusing the program Phaser by first placing the truncated lysozyme(RFZ=3.74; TFZ=3.65) followed by the rhodopsin model (RFZ=5.2; TFZ=7)(McCoy, Acta Crystallogr D Biol Crystallogr 63, 32 (2007)). In order tooptimize placement of the receptor each of the seven helices was refinedindependently by rigid body maximum likelihood refinement as implementedin Refmac (Initial Rwork/Rfree=0.50/0.51) (Murshudov, et al, ActaCrystallogr D D53, 240 (1997)).

Initial rounds of refinement were carried out using restrainedparameters in Refmac. Model rebuilding was performed in Coot utilizing 2Fo-Fc sigma-A weighted maps, as well as density modified maps calculatedusing Resolve prime-and-switch phasing which reduces model biasintroduced by model based phasing methods (Terwilliger, Acta CrystallogrD D56, 965 (2000)). The Resolve calculated maps were superior to thesigma-A weighted ones in that more of the main chain density could betraced. Density for the bound ligand was visible early in the refinementbut was not modeled immediately to allow an unbiased assessment of thephase quality through the improvement of the signal/noise of theobserved ligand density. The structure quality is excellent (Table 3),with strong electron density in particular observed in the ligandbinding site (FIG. 7), cholesterol binding sites (FIG. 8A), and theproline helix kinks (FIG. 8B).

TABLE 3 Data collection and refinement statistics β₂AR-T4L Datacollection (APS GM/CA CAT 23ID-B, 10 μm beam)* Space group C2 Celldimensions a, b, c (Å) 106.32, 169.24, 40.15 β (°) 105.62 No. ofreflections processed 245,571 No. unique reflections 26,574 Resolution(Å) 50-2.4 (2.5-2.4) R_(sym) 12.7 (67.8) Mean I/σ(I) 9.6 (2.2)Completeness (%) 99.5 (99.1) Redundancy 9.4 (4.8) Refinement* Resolution(Å) 20-2.4 (2.46-2.4) No. reflections (test set) 25,247 (1,310)R_(work)/R_(free) 19.8 (27.0)/23.2 (30.1) No. atoms 3,805 Protein 3,544Ions, lipids, ligand and other 213 Water 48 Overall B-values (Å²) 82β₂AR 77 T4-Lysozyme 75 Carazolol 55 Lipid 100 R.m.s deviations Bondlengths (Å) 0.013 Bond angles (°) 1.5 Ramachandran plot statistics (%)(excl. Gly, Pro): Most favored regions 94.8 Additionally allowed regions5.0 Generously allowed regions 0.2 Disallowed regions 0 *Highestresolution shell is shown in parenthesis. R_(sym) = Σ_(hkl)|I(hkl) −<I(hkl)>|/Σ_(hkl)(hkl), where <I(hkl)> is the mean of the symmetryequivalent reflections of I(hkl).

Analysis of β2A Receptor Topology

The final model of β2AR-T4L includes 442 amino acids. β2AR-T4L wastreated with iodoacetamide during purification to eliminate free thiols.The model includes a palmitic acid covalently bound to Cys341 (GPCRs arefrequently post-translationally modified with palmitoylate on cysteineresidues at the C-terminal tail) and an acetamide molecule bound toCys265^(6.27). Throughout the description, residues are designated bytheir position within the β2AR sequence and their Ballesteros-Weinsteindesignation as a superscript where applicable. Ballesteros-Weinsteinnumbering is used throughout the text as superscripts to the proteinnumbering. Within each helix is a single most conserved residue amongthe class A GPCRs. This residue is designated x.50 where x is the numberof the transmembrane helix. All other residues on that helix arenumbered relative to this conserved position. The model also includesone carazolol molecule, three cholesterol molecules, two sulfate ionsand two butanediol molecules that interact with β2AR. There are alsofour sulfate ions, a putative disaccharide (modeled as maltose) and amolecule of PEG 400 bound to T4L. For β2AR, excellent electron densityis observed for residues 29-342, including the ligand carazolol and thetwo disulfide bonds Cys106^(3.25)-Cys191^(5.36) andCys184^(4.76)-Cys190^(5.29). The palmitic acid at Cys341 is clearlyvisible in Fo-Fc omit maps; however, the quality of the electron densityis lower than for the rest of the receptor. The N-terminus (residues 1to 28) and the majority of the C-terminus (residues 343 to 365) aredisordered and not visible in the structure.

The β2AR has a fold composed of seven transmembrane helices forming ahelical bundle (FIG. 9A). The residues that make up the helices (I toVII) in β2AR are as follows: helix 129^(1.28) to 60^(1.59), helix II672.38 to 96^(2.67), helix III 103^(3.22) to 136^(3.55), helix IV147^(4.39) to 171^(4.63), helix V 197^(5.36) to 229^(5.68), helix VI267^(6.29) to 298^(6.60), and helix VII 305^(7.32) to 328^(7.55). Theresidues forming the intracellular loops (ICL) and extracellular loops(ECL) of β2AR are: ICL1 61^(1.66) to 66²³⁷, ECL1 97^(2.68) to102^(3.21), ICL2 137^(3.56) to 146^(4.38), ECL2 172^(4.64) to196^(5.35), ICL3 230^(5.69) to 266^(6.28) (residues 231 to 262 arereplaced by T4-lysozyme residues 2 to 161), and ECL3 299^(6.61) to304^(7.31). Helices II, V, VI and VII each have a proline-induced kinkat conserved positions along the span of the transmembrane segments.These kinks are thought to enable the structural rearrangements requiredfor activation of G protein effectors (Yohannan et al., Proc Natl AcadSci U S A 101, 959 (2004)). In addition to the seven membrane spanninghelices, β2AR possesses two other helical segments: helix VIII, which isbelieved to be common to all rhodopsin-like GPCRs (Katragadda et al.,Biochim Biophys Acta 1663, 74 (2004)), and an unexpected, short helicalsegment in the middle of ECL2, which is not present in rhodopsin, andwas not predicted by computational secondary structure analysis (FIG.9A).

In the β2AR-T4L construct, T4L is fused to the truncated cytoplasmicends of helices V and VI. In the crystal structure, the T4L moiety istilted slightly away from the center axis of β2AR drawn normal to themembrane (FIG. 9B). As a result, interactions between T4L and β2AR areminimal, with only 400A2 of surface area buried between them. Theintramolecular contacts between T4L and β2AR include salt bridgesbetween the side chains of T4L-Asp159 and the side-chain amine ofβ2AR-Lys227^(5.66) (distance 3.4 Å) and between the guanidinium group ofT4L-Arg8 with the side-chain carboxyl of β2AR-Glu268^(6.30) on helix VI(distance 3.2 Å) (FIG. 9C, Table 4). The latter interaction isnoteworthy because it differs from rhodopsin where Glu6.30 forms anionic bond with Arg3.50 of the conserved D(E)RY motif. This interactionis postulated to be important for maintaining rhodopsin in the inactivestate, but the charged groups of the two residues [Arg131^(3.50) (NH1)and Glu268^(6.30) (0E1)] are 10 Å apart in the β2AR-T4L structure. Theremainder of the lysozyme molecule provides important crystal packinginteractions, but does not appear to influence significantly thereceptor structure.

TABLE 4 Direct contacts between β₂AR and T4L van der Waals Contacts β₂ARatom T4 Lysozyme Leu230^(5.69) Trp 158 Lys263^(6.25) Asp159Cys265^(6.27) Ile9 Leu266^(6.28) Ile9 Leu266^(6.28) Glu5 Hydrogen Bondand Salt Bridge Contacts β₂AR atom T4 Lysozyme Distance (Å)Lys227^(5.66) (NZ) Asp159 (OD1) 3.4 Gln229^(5.68) (O) Asn2 (N) 3.1Gln229^(5.68) (O) Asn2 (ND2) 3.2 Gln268^(6.30) (OE2) Arg8 (NH2) 3.2Covalent bonds β₂AR atom T4 Lysozyme Leu230^(5.69) Asn2 Lys263^(6.25)Tyr161

Crystal Packing Interactions

The β2AR-T4L protein is packed in a C-centered monoclinic lattice withone molecule per asymmetric unit (FIG. 10A). Membrane protein generallycan form two types of crystal packing: Type I represents stacks of twodimensional crystals ordered in the third dimension via interactions ofhydrophilic parts of membrane proteins. Type II crystals are composed ofmembrane proteins whose hydrophobic part is shielded by a detergentmicelle and all crystal contacts are formed through hydrophilic, solventexposed parts of protein molecules. As observed in all previous lipidicmesophase grown crystals (Schwartz, et al., Annu Rev Pharmacol Toxicol46, 481 (2006)), the β2AR-T4L crystals adopt Type I packing, featuring amultilayered arrangement in accordance with proposed crystallizationmechanism (Caffrey, Curr Opin Struct Biol 10, 486 (2000); P. Nollert, etal., FEBS Lett 504, 179 (2001)). Within each layer, protein moleculesform arrays of parallel, symmetry-related dimers. There are fourdistinct crystal-packing interactions within each layer, three of whichare mediated by T4L. The fourth interaction in the array is between tworeceptor molecules related by a crystallographic two-fold rotation axis.This is the sole interaction between symmetry-related receptors, and ismediated primarily by ordered lipids consisting of six cholesterol andtwo palmitic acid molecules, the latter being covalently attached toCys341 in the C-terminal portion of the receptor (O'Dowd et al., J BiolChem 264, 7564 (1989)) (FIG. 10B). These eight lipid molecules form atwo-fold symmetric sheet between receptors. The only directreceptor-receptor contact involves a 2.7 Å pair of ionic interactionsbetween the charged amine group of Lys60^(1.59) in helix I and thecarboxylate of Glu³³⁸ in helix VIII from the symmetry-related receptor.Remarkably, of the 515 Å2 buried at the receptor symmetry interface, 73%of the crystal contact surface area is mediated by ordered lipid, whileonly 27% is contributed by protein-protein contacts. The stackinginteractions between layers are formed between T4L and extracellularloops ECL2 and ECL3 of the receptor (FIG. 10A). It is unlikely thatthese contacts affect the orientation of these loops due to the smallsize of ECL3 and the rigid architecture of ECL2.

Lipid Mediated Receptor Association

Many GPCRs including β2AR are thought to exist as dimers in the plasmamembrane, although the location of the dimer interface and thefunctional significance of dimerization is not clear (Milligan, MolPharmacol 66, 1 (2004)). The observation of ordered lipids in the helixI and VIII interface between two symmetry related molecules suggests theassociation is physiologically relevant (Angers, et al., Proc Natl AcadSci U S A 97, 3684 (2000); Javitch, Mol Pharmacol 66, 1077 (2004);Mercier, et al., J Biol Chem 277, 44925 (2002)). Associations betweenthe equivalent regions of rhodopsin have been found in crystalstructures (Salom et al., Proc Natl Acad Sci U S A 103, 16123 (2006);Schertler, Curr Opin Struct Biol 15, 408 (2005)) (FIG. 10C).

The role of cholesterol in the physiologic function of β2AR is welldocumented. Depletion of cholesterol from the membranes of neonatalcardiac myocytes alters the signaling behavior of endogenous β2AR(Xiang, et al., J Biol Chem 277, 34280 (2002)). In untreated cells,activation of β2AR results in sequential coupling to the G proteins Gsand Gi, producing a biphasic effect on myocyte contraction rate. Upondepletion of cholesterol, the β2AR couples more strongly to Gs. Thiseffect may be due to a role of cholesterol in regulating interactionsbetween the β2AR and G proteins, or an effect of cholesterol on β2ARdimerization. The β2AR couples efficiently to Gs as a monomer(Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), so cholesterolmediated association (dimerization) may reduce the efficiency of β2ARcoupling to Gs. The effects of cholesterol depletion on β2AR signalingmay also be a secondary effect of altering subcellular signalingcompartments. There is evidence that cells may concentrate signalingmolecules, such as GPCRs and their cognate G proteins, by way ofmembrane microdomains or compartments, such as caveolae (Ostrom, et al.,Br J Pharmacol 143, 235 (September, 2004)). This compartmentalizationmay be a major regulator of receptor-effector coupling. Thus, theimportance of cholesterol in forming the observed crystallographicassociation is consistent with its role in β2AR signaling.

Electrostatic Charge Distribution

Electrostatic charge distribution was calculated using APBS (Gether,Endocr Rev 21, 90 (2000)) and mapped onto a molecular surfacerepresentation of β2AR. The analysis reveals three polarized areaswithin the molecule (FIG. 11A). First, the cytoplasmic face of thereceptor is involved in G protein interaction and carries a net positivecharge even in the absence of ICL3, which also has a predicted overallpositive charge (FIG. 11B). The second site is an electrostaticallynegative region located within the membrane between helices III, IV andV potentially exposed to the lipid alkyl chains, which is unexpected asthe burial of charge within the plasma membrane is thermodynamicallyunfavorable. A glutamate residue at position 122^(3.41) may partiallyaccount for the observed charge distribution. Finally, the binding sitecleft is negatively charged and exposed to solvent by an unusual ECL2architecture and lack of N-terminal interactions. This negative chargemay facilitate ligand binding through electrostatic funneling ofpositively charged catecholamines (FIG. 11B).

Extracellular Region

The ECLs and amino termini of GPCRs, together with the extracellularhalves of the transmembrane helices, are believed to define theligand-binding site of each receptor (Angers et al., Proc Natl Acad SciU S A 97, 3684 (2000)). Therefore, the ECLs play an important role inthe overall pharmacology of any particular receptor. In general, smallmolecule ligands are thought to bind deeper within the space created bythe transmembrane domain helices, whereas larger ligands such aspeptides bind closer to the membrane surface near the ECLs (Ji, et al.,J Biol Chem 273, 17299 (1998); Gether, Endocr Rev 21, 90 (2000)).Mutagenesis studies suggest that the β2AR binds its ligand deep withinthe transmembrane helix bundle, which may be related to the observationthat the extracellular regions have a rather simple structure with shortloops connecting transmembrane helices II and III, and VI and VII (FIG.12A). ECL2, which links helices IV and V, has a somewhat more extensivearchitecture that is unanticipated. In contrast to the buried, β-sheetstructure of this loop in rhodopsin (FIG. 12B), ECL2 in β2AR is moreexposed to the solvent and contains an extra helical segment.Additionally, there is an intra-loop disulfide bond betweenCys184^(4.76) and Cys190^(5.29) that may help stabilize the more exposedECL2. A second disulfide bond between Cys191^(5.30) and Cys106^(3.25) inhelix III effectively ties ECL2 to the transmembrane core (Noda, et al.,J Biol Chem 269, 6743 (1994)). The distal portion of ECL2 makes closecontacts with ECL1 and contains a glycosylation site at Asn187^(5.26)(Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), which may serveto mask a grouping of aromatic residues on ECL1; in this construct,Asn1875.26 has been mutated to glutamate to aid in crystallization.

Electron density corresponding to the N-terminus was not apparent in themaps and, therefore, residues 1-28 are not included in the model. Thisdisorder contrasts with rhodopsin, in which the N-terminus interactsextensively with the ECLs, forming a small four-strand β-sheet inconjunction with ECL2. This sheet structure forms a cap that effectivelyisolates the retinal binding site in a hydrophobic pocket (FIG. 13B).The lack of interactions between the N-terminus of β2AR and ECL2 furtherenables diffusible ligand access to the binding site. However acompletely disordered N-terminus may be an artifact induced by thepresence of the N-terminal Flag tag which carries an overall positivecharge and may disrupt N-terminal interactions.

The short helical region on ECL2 adds a rigid structural element that,along with the two disulfide bonds, constrains the loop to a small rangeof conformations and helps stabilize the receptor by linking threetransmembrane helices (FIG. 13A). This rigid conformation may help tostabilize the core of the receptor and lock ECL2 in a conformation thatdoes not hinder access to the binding pocket.

Ligand Binding Site and Comparison to Rhodopsin

Carazolol is a partial inverse agonist that binds with picomolaraffinity to β₂AR-T4L producing a reduction of the basal activity of thereceptor. The crystal structure reveals extensive interactions betweenthe receptor and carazolol that position the carbazole moiety adjacentto Phe289^(6.51), Phe290^(6.52), and Trp286^(6.48) (FIG. 13A, FIG. 7,and Table 5). In contrast, cis-retinal is a full inverse agonistcovalently bound to rhodopsin, which suppresses all activity towardstransducin (Palczewski, Annu Rev Biochem 75, 743 (2006)). Carazolol andretinal occupy similar spaces in their respective receptors, withsignificant overlap of the non-aromatic regions of carazolol. However,the β-ionone ring of retinal extends deep into the binding pocket ofrhodopsin and contacts residues on helix V and VI, where it issandwiched between Phe212^(5.47) and Tyr268^(6.51), and interacts withthe highly conserved Trp265^(6.48) (FIG. 13B). It has been proposed thatchanges in the rotamer of Trp265^(6.48) occur upon activation ofrhodopsin and related family members, and constitutes the “toggleswitch” for receptor activation (Schwartz, et al., Annu Rev PharmacolToxicol 46, 481 (2006)). Accordingly, the interactions betweenc/s-retinal and Trp265^(6.48) are likely to contribute to the absence ofbasal activity in rhodopsin. Carazolol does not interact directly withthe toggle switch on helix VI, however it lowers the basal activity ofthe receptor, and may do so by interacting with Phe289^(6.51) andPhe290^(6.52), which form an extended aromatic network surrounding thehighly conserved Trp286^(6.48). As a result, Trp286^(6.48) adopts therotamer associated with the inactive state. Thus, the steric constraintsimposed by Phe290^(6.52) appear to structurally mimic the interaction ofthe β-ionone ring of retinal with the conserved Trp265^(6.48) andPhe212^(5.47) on rhodopsin (Shi et al., J Biol Chem 277, 40989 (2002))(FIG. 13C).

TABLE 5 Direct contacts between β₂AR and carazolol. β₂AR atom CarazololDistance (Å) Hydrogen Bond and Salt Bridge Contacts Asp113^(3.32) (OD2)N19 2.9 Asp113^(3.32) (OD1) O17 2.6 Ser203^(5.42) (OG) N7 3.2Asn312^(7.39) (ND2) O17 2.9 Asn312^(7.39) (OD1) N19 2.9 Tyr316^(7.43)(OH) N19 3.4 Hydrophobic and Aromatic Interactions (closest distance foreach residue, <4 Å) Trp109^(3.28) (CH2) C21 3.8 Val114^(3.33) (CG1) C113.9 Val117^(3.36) (CG1) C12 4.0 Thr118^(3.37) (OG1) C11 3.9Phe193^(5.32) (CE2) C6 3.5 Tyr199^(5.38) (CE2) C2 3.9 Ser207^(5.46) (CB)C10 3.6 Trp286^(6.48) (CH2) O17 3.4 Phe289^(6.51) (CE2) O14 3.7Phe290^(6.52) (CZ) C12 3.5 Asn293^(6.55) (ND2) C5 3.6 Tyr308^(7.35) (OH)C6 3.6

Structural Alignment and Helix Bundle Reorganization

It has long been thought that class A GPCRs share a similar architecturedue to their predicted seven transmembrane helical bundles and sequenceconservation within the membrane spanning regions (Lefkowitz, Nat CellBiol 2, E133 (2000)). We aligned the structure of β2AR-T4L to highestresolution structure of rhodopsin (PDB ID Code 1U19) to evaluate thesimilarities and differences in ligand binding modes. We used differencedistance matrices to select non-divergent areas between the twostructures that align to reveal the differences in helix orientationbetween β2AR-T4L and rhodopsin. For the alignment, residues on β2AR werealigned to equivalent residues on Rhodopsin, respectively: 43-59 to47-63; 67-95 to 71-99; 122-135 to 126-139; 285-296 to 264-275.

Relative to rhodopsin, the following helical shifts are seen inβ2AR-T4L: the extracellular portions of helices I and III angle awayfrom the center of the receptor, helix IV is translated away from thecenter of the receptor, helix V is translated closer to the center ofthe receptor and helix VI angles away from the receptor on thecytoplasmic end (FIG. 14). The largest difference is in helix I, whichlacks a proline-induced kink found in rhodopsin and is comparativelystraight. The angle between the rhodopsin and β2AR positions of helix Iis approximately 18° with a shift of 7 Å at the apex on theextracellular face. This structural difference may arise from the needfor an accessible binding site in β2AR, which is provided in part by alack of interactions between the N-terminus and extracellular loopsegments. In contrast the N-terminal region in rhodopsin occludes theretinal-binding site through extensive interactions with theextracellular loops (FIG. 12B). Helix V of β2AR is closer to the bindingpocket by approximately 3.5 Å on average and its lumenal end is angledmore towards helix VI. Helix IV of β2AR is further from the bindingsite, possibly to remove steric clashes resulting from the modifiedposition of helix V (FIG. 14B, 14C). Helix III pivots further from thebinding site about a fulcrum located close to the cytoplasmic end (FIG.14C). The angle formed between rhodopsin helix III and the β2AR helixIII is approximately 7°, yielding a 4 Å displacement out of the bindingpocket at the cytoplasmic end of the helix. Helix VI is positionedfurther from the center of the receptor at the cytoplasmic end ascompared to rhodopsin, which is caused by a slight difference in theangle about the proline-induced kink in the helix (FIG. 14C).

The ligand-binding pocket is formed by both structurally conserved anddivergent helices as compared to rhodopsin (FIG. 14D). Helices III and Vare two of the most conformationally shifted helices and contain thecanonical catecholamine binding residues associated with activation ofadrenergic family of receptors (Strader et al., J Biol Chem 263, 10267(1988); Strader, et al., J Biol Chem 264, 13572 (1989); Liapakis et al.,J Biol Chem 275, 37779 (2000)). The comparison with rhodopsin shows thatthe structurally conserved helices provide a common core presentthroughout the class A GPCRs, whereas the variable helices conferbinding site plasticity with a resulting architecture capable of bindinga large spectrum of ligands.

Comparison to Rhodopsin-Based GPCR Models

Since the determination of the inactive dark-state rhodopsin structure(Palczewski et al., Science 289, 739 (2000)), a number of homologymodels of other class A GPCRs have been reported (Bissantz, et al.,Proteins 50, 5 (2003); Fano, et al., J Chem Inf Model 46, 1223 (2006);Hobrath, et al., J Med Chem 49, 4470 (2006); Nowak, et al., J Med Chem49, 205 (2006); Zhang, et al., PLoS ComputBiol 2, e13 (2006)).Typically, homology models start by alignment of so-called fingerprintmotifs that are common among the family. These fingerprint motifs areextrapolated to assign coordinates for the entire helical bundle. Loopregions are either ignored or modeled based on databases of loopconformations depending on the application (Bissantz, et. al, Proteins50, 5 (2003)). A number of models exist for β2AR, some of which havebeen improved upon with supporting biochemical data (Bissantz, et. al,Proteins 50, 5 (2003); Zhang, et al., PLoS ComputBiol 2, e13 (2006);Freddolino et al., Proc Natl Acad Sci U S A 101, 2736 (2004); Furse, etal., J Med Chem 46, 4450 (2003); Gouldson et al., Proteins 56, 67(2004)). When compared to the β2AR structure reported here (according tothe methods described above in this Example), however, all of thesemodels were more similar to rhodopsin, as were models for otherreceptors (e.g. dopamine, muscarinic, and chemokine). This highlights ageneral shortcoming in homology models generated from a singlestructural template. The structural divergence between β2AR andrhodopsin would be quite difficult to predict accurately using onlyrhodopsin as a template.

Example 4 Structural Insights into β₂ Adrenergic Receptor Function

Methods

Molecular Biology for Generation of Mammalian and Sf9 ExpressionConstructs.

The insect cell expression plasmid that was used as a template formodification of the human β2AR gene has been described previously (Yaoet al., Nat Chem Biol 2, 417 (2006)): the wild-type coding sequence ofthe human β2AR (starting at Gly2) was cloned into the pFastbac1 Sf-9expression vector (Invitrogen) with the HA signal sequence followed bythe Flag epitope tag at the amino terminus and the third glycosylationsite mutated as N187E. Using this template, a TAA stop codon was placedbetween Gly365 and Tyr366, terminating translation without the 48C-terminal residues of the wild-type β2AR (β2AR365″). A synthetic DNAcassette encoding the T4 Lysozyme (WT*-C54T, C97A) protein was made byoverlapping extension PCR of 50-base oligonucleotides. This cassette wasamplified and inserted into the β2AR365 construct between Ile233^(5.72)and Arg260^(6.22) (FIG. 21A), using the Quickchange Multi protocol(Stratagene). The corresponding mammalian cell expression plasmid wasmade by amplifying the entire fusion gene and cloning it into pcDNA3(Invitrogen). Further deletions in the Sf9 and mammalian cell constructswere made using appropriate synthetic oligonucleotides in theQuickchange Multi protocol (Stratagene). All constructs were confirmedby sequencing.

HEK293 Cell Staining and Immunofluorescence Staining.

HEK293 cells were cultured on plastic dishes at 37° C. with 5% CO2 inDulbecco's modified Eagle's medium (Cellgro) with 5% fetal bovine serum.For an individual expression experiment, cells at confluency were split,and approximately 100,000 cells were used to seed glass cover slips inthe same medium. After 2 d, cells were transfected with the addition of1 μg of a given pcDNA3-receptor plasmid and 3 μl of Fugene 6 reagent(Roche). 48 h after transfection, cells were washed with PBS, fixed with4% paraformaldehyde, blocked with PBS+2% goat serum, permeabilized withPBS+2% goat serum+0.5% Nonidet P-40 (Sigma), stained withAlexa488-conjugated M1 anti-FLAG antibody (for receptor) plus DAPI(nuclear) in blocking buffer, and washed with blocking buffer. Coverslips were mounted on microscope slides with Vectashield (Vector Labs)and dried overnight. Staining was visualized with an Axioplan 2fluorescence imaging system, using a 63× objective and either green(Alexa488/FITC) or blue (DAPI/Hoechst) filter sets. A plasmidpcDNA3-β1AR, expressing an N-terminal FLAG-tagged β1 adrenergicreceptor, was used as a positive control for cell-surface staining.Empty pcDNA3 was used as a negative control to assess backgroundstaining.

Expression and Purification of β₂AR-T4L from Baculovirus-Infected Sf9Cells.

Recombinant baculovirus was made from pFastbac1-β2AR-T4L using theBac-to-Bac system (Invitrogen), as described previously (Yao et al., NatChem Biol 2, 417 (2006)). The β2AR-T4L protein was expressed in Sf9insect cells infected with this baculovirus, and solubilized accordingto previously described methods (Kobilka, Anal Biochem 231, 269 (1995)).Dodecylmaltoside-solubilized receptor with the N-terminal FLAG epitope(DYKDDDA) (SEQ ID NO: 1) was purified by M1 antibody affinitychromatography (Sigma), treated with TCEP/iodoacetamide, and furtherpurified by alprenolol-Sepharose chromatography (Kobilka, Anal Biochem231, 269 (1995)) to isolate only functional GPCR. Elutedalprenolol-bound receptor was re-bound to M1 FLAG resin, and ligandexchange with 30 μM carazolol was performed on the column. β2AR-T4L waseluted from this final column with 0.2 mg/ml FLAG peptide in HLS buffer(0.1% dodecylmaltoside, 20 mM Hepes, 100 mM NaCl, pH 7.5) plus 30 μMcarazolol and 5 mM EDTA. N-linked glycolsylations were removed bytreatment with PNGaseF (NEB). Protein was concentrated from ˜5 mg/ml to50 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator(Vivascience), and dialyzed against HLS buffer plus 10 μM carazolol.

Binding Measurements on Wild-Type β₂AR and β₂AR-T4L from Membranes.

Membrane preparation from baculovirus-infected Sf9 cells was performedas described previously (Swaminath, et al., Mol Pharmacol 61, 65(2002)). For each binding reaction, membranes containing 0.7 μg totalmembrane protein were used. Saturation binding of [³H]-dihydroalprenolol(DHA) was measured by incubating membranes resuspended in 500 μl bindingbuffer (75 mM Tris, 12.5 mM MgCl₂, 1 mM EDTA, pH 7.4, supplemented with0.4 mg/ml BSA) with 12 different concentrations of [³H]DHA (PerkinElmer) between 20 pM and 10 nM. After 1 h incubation with shaking at 230rpm, membranes were filtered from the binding reactions with a Brandelharvester, washed with binding buffer, and measured for bound [³H]DHAwith a Beckman LS6000 scintillation counter. Non-specific binding wasassessed by performing identical reactions in the presence of 1 μMalprenolol. For competition binding, membranes resuspended in 500 μlbinding buffer were incubated with 0.5 nM [³H]DHA plus increasingconcentrations of the competing ligand (all compounds were purchasedfrom Sigma). For (−)-isoproterenol and (−)-epinephrine, concentrationswere 100 pM-1 mM, each increasing by a factor of 10. For salbutamol,concentrations were 1 nM-10 mM. For ICI-118,551 and formoterol,concentrations were 1 pM-10 μM. Non-specific binding was measured byusing 1 μM unlabeled alprenolol as competing ligand. Each data point inthe curves in FIGS. 2A and S1 represents the mean of three separateexperiments, each done in triplicate. Binding data were analyzed bynonlinear regression analysis using Graphpad Prism. The values for K_(d)of [³H]DHA and K_(i) of other ligands are shown in Table 6.

TABLE 6 Saturation Binding [³H]DHA K_(d) ± SE (nM) Bmax (pmol/mg) β₂AR0.161 ± 0.012 30.0 ± 0.5 β₂AR-T4L 0.180 ± 0.016 21.6 ± 0.5 CompetitionBinding K_(i) [S.E. interval] K_(i) [S.E. interval] Ligand for β₂AR (nM)for β₂AR-T4L (nM) (−)-isoproteronol 50.6 [48.9-52.3] 15.7 [15.2-16.2](−)-epinephrine 175 [163-188]  56.0 [52.8-59.4] salbutamol 728[708-750]  307 [291-323]  ICI-118,551  0.617 [0.570-0.668]  0.626[0.591-0.662] formoterol 3.60 [3.39-3.83] 1.68 [1.55-1.81] Bindingaffinities of different ligands for the wild-type β₂AR and the fusionprotein β₂AR-T4L. The saturation and competition binding curves shown inFIG. 22 were fit to theoretical saturation and one-site competitionbinding models, using the program Graphpad Prism. K_(i) values werecalculated using the Cheng-Prusoff equation: K_(i) = IC₅₀/(1 +[ligand]/K_(d)).

Bimane Fluorescence Experiments on Purified, Detergent-SolubilizedReceptors

β₂AR-T4L and β₂AR365 were purified as described above, with twoimportant differences. First, prior to iodoacetamide treatment,FLAG-pure receptor at 2.5 μM (measured by soluble [³H]DHA binding) wasincubated with 5 μM monobromobimane for 1 h at 4° C. Second, afterbinding the bimane-labeled alprenolol-Sepharose-purified receptor to M1antibody resin, the column was washed extensively with ligand-freebuffer before elution. Based on previous precedent (Ghanouni, et al.,Proc Natl Acad Sci U S A 98, 5997 (2001)), this protocol is expected totarget primarily Cys265^(6.27) for fluorophore derivitization.Fluorescence spectroscopy was performed on a Spex FluoroMax-3spectrofluorometer (Jobin Yvon Inc.) with photon-counting mode, using anexcitation and emission bandpass of 5 nm. All experiments were done at25° C. For emission scans, we set excitation at 350 nm and measuredemission from 417 to 530 nm with an integration time of 1.0 s nm⁻¹. Todetermine the effect of ligands, spectra were measured after 15 minincubation with different compounds (at saturating concentrations:[(−)-isoproterenol]=100 μM; [ICI-118,551]=10 μM; [salbutamol]=500 nM).Fluorescence intensity was corrected for background fluorescence frombuffer and ligands in all experiments. The curves shown in FIG. 22B areeach the average of triplicate experiments performed in parallel.λ_(max) values and intensity changes for β₂AR-T4L and β₂AR365, eachincubated with different ligands, are tabulated in Table 7.

TABLE 7 λmax ± SD for λmax ± SD for Ligand (β₂AR365 (nm) β₂AR-T4L (nm)none 448 ± 2 447 ± 2 (−)-isoproteronol 453 ± 2 455 ± 2 ICI-118,551 447 ±1 446 ± 1 salbutamol 449 ± 1 449 ± 1 Intensity atλmax_(Ligand)/Intensity at λmax_(none) Ligand β₂AR365 β₂AR-T4L(−)-isoproteronol 0.758 ± 0.007 0.824 ± 0.006 ICI-118,551 1.013 ± 0.0081.028 ± 0.008 salbutamol 0.950 ± 0.013 0.928 ± 0.009 Bimane fluorescenceresponses for unliganded β₂AR365 and β₂AR-T4L, incubated for 15 min withdifferent ligands. Top panel shows the λ_(max) for fluorescence emissionspectra (excitation at 350 nm and emission from 417 to 530 nm) collectedafter 15 min incubation with ligand. Each value is mean ± standarddeviation for triplicate experiments performed in parallel. Bottom panelshows the change in fluorescence intensity after incubation with ligand,represented as the ratio of Intensity at λmax of the ligand to Intensityat λ_(max) of the control no ligand (“none”) response.

Comparing the Proteolytic Stability of Unliganded β₂AR and β₂AR-T4L.

The limited trypsin proteolysis protocol was adapted from Jiang et al.,Biochemistry 44, 1163 (2005). Carazolol-bound β₂AR-T4L or wild-type β₂AR(each at 30 mg/ml) were diluted 10-fold into HLS buffer (see above) andTPCK-trypsin was added at a 1:1000 ratio (wt:wt). The digests wereincubated at room temperature. At various time points, aliquots wereremoved and flash frozen on dry ice/ethanol. After the last aliquot wasremoved, all samples were thawed, and an equal volume of 10% SDS/PAGEloading buffer was added to each. Samples were then analyzed byelectrophoresis on 12% polyacrylamide gels, followed by staining withCoomassie blue (FIG. 16).

Comparing the Stability of Unliganded β₂AR and β₂AR-T4L

Unliganded β2AR365 and β2AR-T4L were each purified as described abovefor the bimane experiments. 200 μl 0.02 mg/ml receptor in HLS buffer wasincubated at 37° C. on a heating block. At the time points indicated inFIG. 17, samples were briefly spun and gently vortexed and 16.5 μl wasremoved and diluted 18.2-fold in HLS (300 μl total). Then 4×5 μl wasremoved for determination of total binding and 2×5 μl was removed fornonspecific binding. To measure soluble binding, 5 μl diluted receptorwas added to 105 μl HLS (400-fold final dilution of receptor) containing10 nM [3H]DHA±10 μM cold alprenolol. Reactions were incubated 30 min atRT, then on ice until processing. 100 μl of each reaction was applied toa 1 ml G50 column to separate protein from residual unbound [3H]DHA, andreceptor was eluted using 1.1 ml ice-cold HLS. Bound [3H]DHA wasquantified on a Beckman LS6000 scintillation counter.

Carazolol Dissociation from the “Wild-Type” Receptor β₂AR365

β2AR365 was purified with carazolol bound, according to the protocoldescribed above for β2ART4L. Carazolol-bound receptor (at approximately50 μM concentration) was dialyzed in the dark against 1L dialysis buffer(20 mM HEPES pH7.5, 100 mM NaCl, 0.1% dodecylmaltoside, 300 micromolaralprenolol) at room temperature with stirring. At indicated time points,two samples were removed from the parafilm-sealed open-ended dialysischamber, diluted into fresh dialysis buffer, and carazolol emissionspectra were obtained on a Spex FluoroMax spectrofluorometer (usingexcitation at 330 nm and emission from 335 to 400 nm). As internalstandards for every time point, samples were removed for determinationof protein concentration using the Bio-Rad Protein DC kit (FIG. 19).

CAM and UCM Mutants

The CAMs (constitutively active mutants) described in the literaturethat are the basis for FIG. 26A and the associated discussion are: L124A(Tao, et al., Mol Endocrinol 14, 1272 (2000)), C116F (Zuscik, et al., JBiol Chem 273, 3401 (1998)), D130A (Rasmussen et al., Mol Pharmacol 56,175 (1999)), L272C (Jensen et al., J Biol Chem 276, 9279 (2001)), andC285T (Shi et al., J Biol Chem 277, 40989 (2002)). The UCMs (uncouplingmutations) from the literature that were used for FIG. 26C are: D79N(Chung, et al., J Biol Chem 263, 4052 (1988)), F139A (Moro, et al., JBiol Chem 269, 6651 (1994)), T1641 (Green, et al., J Biol Chem 268,23116 (1993)), N318K (Strader et al., Proc Natl Acad Sci U S A 84, 4384(1987)), N322A (Barak, et al., Biochemistry 34, 15407 (1995)), P323A(Barak, et al., Biochemistry 34, 15407 (1995)), Y326A (Barak, et al.,Biochemistry 34, 15407 (1995)), L339A (Gabilondo et al., Proc Natl AcadSci U S A 94, 12285 (1997)), and L340A (Gabilondo et al., Proc Natl AcadSci U S A 94, 12285 (1997)).

Biochemical and Structural Analysis of β₂AR-T4L

The β₂AR fusion protein in which T4 Lysozyme replaces most of the thirdintracellular loop of the GPCR (“β₂AR-T4L”) retains near-nativepharmacologic properties. The β₂AR-T4L protein was crystallized inlipidic cubic phase, as described in the Examples above, and theresulting 2.4 Å resolution crystal structure reveals the interfacebetween the receptor and the ligand carazolol, a partial inverseagonist. The efficacy of a ligand describes the effect of the ligand onthe functional properties of a GPCR. For purposes of the Examples only,agonists are defined as ligands that fully activate the receptor;partial agonists induce submaximal activation even at saturatingconcentrations; inverse agonists inhibit basal receptor activity, andantagonists have no effect on basal activity, but competitively blockaccess of other ligands. Carazolol, is defined as a partial inverseagonist because it suppresses only 50% of the basal activity of theβ₂AR. Analysis of mutagenesis data in light of the structure clarifiesthe roles of different amino acids in inverse agonist binding, andimplies that rearrangement of the binding pocket accompanies agonistbinding. In addition, the structure reveals how mutations known to causeconstitutive activity or uncoupling of agonist binding and G-proteinactivation are distributed between the ligand-binding pocket and thecytoplasmic surface of the protein, such that changes in side chains dueto interaction with the ligand can be transmitted through the structureto the site of G protein interaction.

Cloning of β₂AR-T4L

DNA encoding the T4L protein (C54T, C97A) (Matsumura, et al., Proc NatlAcad Sci U S A 86, 6562 (1989)) was initially cloned into the human β2ARgene, guided by comparison of ICL3 length and sequence among class AGPCRs (Horn et al., Nucleic Acids Res 31, 294 (2003)): residues234^(5.23)-259^(6.21) of the β2AR were replaced by residues 2-164 of T4L(construct “E3” in FIG. 21A). In addition, the receptor was truncated atposition 365, which aligns approximately with the position of therhodopsin carboxyl terminus. Although these modifications resulted in areceptor that was expressed efficiently in Sf9 cells, furtheroptimization was carried out to reduce the length of the junctionbetween the receptor and the T4L termini, as described in the methodsabove. Several candidate constructs are illustrated in FIG. 21A, andselected immunofluorescence images of transfected, permeabilized HEK293cells are shown in FIG. 21B. Relative to the initial construct, we couldremove three residues from the cytoplasmic end of helix V, threeresidues from the C-terminal end of T4L, and three residues from the Nterminus of helix VI, all without losing significant cell-surfaceexpression. The final construct used for crystallization trials(“β2AR-T4L”) has residues 231^(5.70)-262^(6.24) of the β2AR replaced byamino acids 2-161 of T4L (“1D” in FIG. 21A). Similar reduction offlexibility through minimization of linker length has been important inprevious crystallization studies on soluble fusion proteins (Smyth, etal., Protein Sci 12, 1313 (2003)).

Functional Properties of β₂AR-T4L

We measured saturation binding of [3H]DHA to the β2AR-T4L, as well ascompetition binding of the inverse agonist ICI-118,551 and severalagonists (FIG. 22A, FIG. 15 and Table 6). The results show that β2AR-T4Lhas wild-type affinity for the antagonist [3H]DHA and the inverseagonist ICI-118,551, whereas the affinity for both agonists(isoproterenol, epinephrine, formoterol) and a partial agonist(salbutamol) is two to three-fold higher relative to wild-type β2AR.Higher agonist binding affinity is a property associated withconstitutively active mutants (CAMs) of GPCRs. CAMs of the β2AR alsoexhibit elevated basal, agonist-independent activation of Gs, andtypically have lower expression levels and reduced stability (Gether etal., J Biol Chem 272, 2587 (1997); Rasmussen et al., Mol Pharmacol 56,175 (1999)). β2ART4L exhibits binding properties of a CAM, but itexpresses at levels exceeding 1 mg per liter of Sf9 cell culture, ismore resistant to trypsin proteolysis than the wild-type β2AR (FIG. 16),and retains binding activity in detergent at 37° C. as well as thewild-type receptor (FIG. 17).

β2AR-T4L did not couple to Gs, as expected due to the replacement ofICL3 by T4L. To assess whether the fused protein alters receptorfunction at the level of its ability to undergo conformational changes,we used a covalently attached fluorescent probe as a reporter forligand-induced structural changes. Fluorophores attached atCys265^(6.27), at the cytoplasmic end of helix VI, detectagonist-induced conformational changes that correlate with the efficacyof the agonist towards G protein activation (Ghanouni et al., J BiolChem 276, 24433 (2001); Ghanouni, et al., Proc Natl Acad Sci U S A 98,5997 (2001); Swaminath et al., J Biol Chem 279, 686 (2004); Swaminath etal., J Biol Chem 280, 22165 (2005)). Detergent-solubilized β2AR365(wild-type receptor truncated at 365) and β2AR-T4L were each labeledwith monobromobimane. Addition of the agonist isoproterenol to purifiedβ2AR365 induces a decrease in fluorescence intensity and a shift in λmaxfor the attached bimane probe (FIG. 22B and Table 7). These changes inintensity and λmax are consistent with an agonist-induced increase inpolarity around bimane. A smaller change is observed with the partialagonist salbutamol, while the inverse agonist ICI-118,551 had littleeffect. For the β2AR-T4L, there are subtle differences in the baselinespectrum of the bimane-labeled fusion protein, as might be expected ifthe environment around Cys265^(6.27) is altered by T4L. However, thefull agonist isoproterenol induces a qualitatively similar decrease inintensity and rightward shift in λmax. Thus the presence of the fusedT4L does not prevent agonist-induced conformational changes. The partialagonist salbutamol induced larger responses in β2AR-T4L than wereobserved in wild-type β2AR, and there was a small increase influorescence in response to the inverse agonist 10-118,551. These areproperties observed in CAMs (Gether et al., J Biol Chem 272, 2587(1997); Samama, et al., J Biol Chem 268, 4625 (1993)) and are consistentwith the higher affinities for agonists and partial agonists exhibitedby β2AR-T4L. Therefore, we conclude that the T4L fusion induces apartial constitutively active phenotype in the β2AR, likely caused bychanges at the cytoplasmic ends of helices V and VI.

Comparison Between β₂AR-T4L and β₂AR-Fab Structures

The β₂AR-T4L fusion strategy is validated by comparison of its structureto the structure of wild-type β₂AR complexed with a Fab that recognizesa three dimensional epitope consisting of the amino andcarboxyl-terminal ends of ICL3, determined at an anisotropic resolutionof 3.4 Å/3.7 Å (Rasmussen et al., Nature, 7168:355-6 (2007)). FIG. 23Aillustrates the similarity between the fusion and antibody complexapproaches to β₂AR crystallization, in that both strategies rely onattachment (covalent or non-covalent, respectively) of a soluble proteinpartner between helices V and VI. A major difference between the twostructures is that the extracellular loops and the carazolol ligandcould not be modeled in the β₂AR-Fab complex, whereas these regions areresolved in the structure of β₂AR-T4L. Nonetheless, it is clear that theT4L insertion does not significantly alter the receptor. Superpositionof the two structures (FIG. 18) illustrates that the transmembranehelices of the receptor components are very similar (RMSD=0.8 Å for 154common modeled transmembrane Cα positions, versus 2.3 Å between β₂AR-T4Land the 154 equivalent residues in rhodopsin), especially when themodest resolution of the Fab complex is taken into account.

There is one significant difference between the Fab-complex and chimericreceptor structures that can be attributed to the presence of T4L. Thecytoplasmic end of helix VI is pulled outward as a result of the fusionto the carboxyl terminus of T4L, which alters the packing ofPhe264^(6.26) at the end of helix VI (FIG. 23B). In the Fab-complexβ₂AR, interactions between Phe264^(6.26) and residues in helix V, helixVI, and ICL2 may be important in maintaining the β₂AR in the basalstate. The loss of these packing interactions in β₂AR-T4L couldcontribute to the higher agonist binding affinity characteristic of aCAM.

An unexpected difference between the structure of rhodopsin and theβ₂AR-T4L involves the sequence E/DRY found at the cytoplasmic end ofhelix III in 71% of class A GPCRs. In rhodopsin, Glu134^(3.49) andArg135^(3.50) form a network of hydrogen bond and ionic interactionswith Glu247^(6.36) at the cytoplasmic end of helix VI. Theseinteractions have been referred to as an “ionic lock” that stabilizesthe inactive state of rhodopsin and other class A members (Ballesteroset al., J Biol Chem 276, 29171 (2001)). However, the arrangement of thehomologous residues is significantly different in β₂AR-T4L: Arg131^(3.56) interacts primarily with Asp130^(3.49) and a sulfate ion ratherthan with Glu268^(6.36), and the distance between helix III and helix VIis greater than in rhodopsin (FIG. 23C). The fact that similar ioniclock structures were obtained using two different approaches suggeststhat a broken ionic lock is a genuine feature of the carazolol-boundstate of the receptor.

Ligand Binding to the β₂AR

The β₂AR-T4L fusion protein was purified and crystallized in complexwith the inverse agonist carazolol. Carazolol stabilizes the β₂ARagainst extremes of pH and temperature, perhaps related to its unusuallyhigh binding affinity (K_(d)<0.1 nM) and slow dissociation kinetics(t_(1/2)˜30 h) (FIG. 19). The interactions between carazolol andβ₂AR-T4L are depicted schematically in FIG. 24. The carbazole ringsystem is oriented roughly perpendicular to the plane of the membrane,and the alkylamine chain (atoms 15-22 in the model) is nearly parallelto the heterocycle (FIG. 25A-B). As described in Example 3, above,carazolol was modeled into the electron density as the (S)-(−) isomerdue to the higher affinity of this enantiomer, despite the fact that aracemic mixture of the ligand was used in crystallization. Asp113^(3.32), Tyr316^(7.43), and Asn312^(7.39) present a constellation ofpolar functional groups to the alkylamine and alcohol moieties of theligand, with Asp113^(3.32) and Asn312^(7.39) sidechains forming closecontacts (<3 Å) with O₁₇ and N₁₉ atoms of carazolol (FIGS. 24 and25A-B). Asp 113^(3.32) was one of the first β₂AR residues shown to beimportant for ligand binding; notably the D113N mutation causes completeloss of detectable affinity for antagonists (Strader et al., Proc NatlAcad Sci U S A 84, 4384 (1987)) and a decrease in the potency ofagonists towards cell-based G protein activation by over 4 orders ofmagnitude (Strader et al., J Biol Chem 263, 10267 (1988)). Likewise,mutations of Asn312^(7.39) perturb β₂AR binding to agonists andantagonists: changes to nonpolar amino acids (Ala or Phe) reduceaffinities to undetectable levels, while retention of a polarfunctionality (Thr or Gln) gives partial affinity (Suryanarayana, etal., Mol Pharmacol 44, 111 (1993)). On the opposite end of the ligandnear helix V, N₇ of the carbazole heterocycle forms a hydrogen bond withthe side chain hydroxyl of Ser203^(5.42). Interestingly, mutations ofSer203^(5.42) specifically decrease β₂AR affinity towards catecholamineagonists and aryloxyalkylamine ligands with nitrogen-containingheterocycles such as pindolol (Liapakis et al., J Biol Chem 275, 37779(2000)), and by implication carazolol. Thus, the polar interactionsbetween carazolol and the receptor observed in the crystal structureagree with the known biochemical data. The contribution of Tyr316^(7.43)to antagonist and agonist affinity remains to be tested; this residue isconserved as tyrosine in all sequenced adrenergic receptor genes (Hornet al., Nucleic Acids Res 31, 294 (2003)).

FIG. 25C shows the tight packing between carazolol and surrounding aminoacids that buries 790 Å² of surface area from solvent; specific contactsare depicted schematically in FIG. 24. Notable among the hydrophobicresidues contacting carazolol are Val114^(3.33), Phe290^(6.52), andPhe193^(5.32). The side chain of Val114^(3.33) from helix III makesmultiple contacts with the C₈-C₁₃ ring of the carbazole heterocycle, andPhe290^(6.52) from helix VI forms an edge-to-face aromatic interactionwith the same ring. As a result, these two amino acids form ahydrophobic “sandwich” with the portion of the aryl moiety that iscommon to many adrenergic antagonists. Mutation of Val114^(3.33) toalanine was shown to decrease β₂AR affinity towards the antagonistalprenolol by an order of magnitude, as well as lowering affinity forthe agonist epinephrine 300-fold (P. Chelikani et al., Proc Natl AcadSci U S A 104, 7027 (2007)). Phe193^(5.32) is different from othercarazolol contact residues in that it is located on the ECL2, in thepath of hormone accessibility to the binding pocket. This amino acidcontributes more buried surface area than any other residue to theinterface between β₂AR-T4L and carazolol (see Table 8). Therefore,Phe193^(5.32) is likely to contribute significantly to the energy ofβ₂AR-carazolol complex formation, and the position of this residue onthe extracellular side of the binding site may allow it to act as a gatethat contributes to the unusually slow dissociation of the ligand (FIG.19).

TABLE 8 β₂AR residue Surface area buried (Å²) Trp109^(3.28) 21.4Thr110^(3.29) 5.7 Asp113^(3.32) 19.3 Val114^(3.33) 25.5 Val117^(3.36)8.5 Thr118^(3.37) 1.9 Phe193^(5.32) 51.2 Thr195^(5.34) 7.4 Tyr199^(5.38)7.6 Ala200^(5.39) 10.0 Ser203^(5.42) 9.0 Ser204^(5.43) 4.6 Ser207^(5.46)6.3 Trp286^(6.48) 3.1 Phe289^(6.51) 20.0 Phe290^(6.52) 19.0Phe293^(6.55) 18.7 Tyr308^(7.35) 14.4 Asn312^(7.39) 22.5 Tyr316^(7.43)6.5 Buried surface area contributions at the β₂AR-T4L/carazololinterface. Solvent accessible surface area calculations were done withthe CNS software package (Brunger et al., Acta Crystallogr D BiolCrystallogr 54, 905 (1998)), using a probe radius of 1.4 Å. Buriedsurface area contributions of individual residues were determined bycalculating solvent-accessible surface area per residue for the fullβ₂ART4L/carazolol model, and subtracting these numbers from thecalculated values for the receptor model without carazolol.

Analysis of the binding pocket provides insights into the structuralbasis for pharmacologic selectivity between the β₂AR and closely relatedadrenergic receptors such as the β₁AR. The affinities of these tworeceptors for certain ligands, such as ICI-118,551, betaxolol and RO363(Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)), differ by up to100-fold. Curiously, all of the amino acids in the carazolol bindingpocket are conserved between the β₁AR and β₂AR (see FIG. 20). Themajority of the 94 amino acid differences between the β₁AR and β₂AR arefound in the cytoplasmic and extracellular loops. While residues thatdiffer in the transmembrane segments generally face the lipid bilayer,eight residues lie at the interface between helices and may influencehelix packing. The structural basis for pharmacologic differencesbetween β₁AR and β₂AR must, therefore, arise from amino acid differencesin the entrance to the binding pocket or subtle differences in thepacking of helices. Evidence for the latter comes from chimeric receptorstudies (Frielle, et al., Proc Nad Acad Sci U S A 85, 9494 (1988)) inwhich successive exchange of helices between β₁AR and β₂ARs led to agradual change in affinity for the β₂AR selective ICI-118,551 and theβ₁AR selective betaxolol.

As discussed above, β₂AR-T4L shows CAM-like properties with respect toagonist binding affinities, suggesting that the unliganded β₂AR-T4L mayexist in a more active conformation than the wild type-β₂AR.Nevertheless, as shown in FIG. 22B, β₂AR-T4L can be stabilized in aninactive conformation by an inverse agonist. Since β₂AR-T4L wascrystallized with bound carazolol, a partial inverse agonist, thestructure most likely represents an inactive state. This is consistentwith the similarity of the β₂AR-T4L and β₂AR-Fab5 carazolol-boundstructures. To assess whether conformational changes are required toaccommodate catecholamines, a model of isoproterenol was placed in thebinding site such that common atoms (16-22 in FIG. 24) were superimposedonto the analogous carazolol coordinates in the crystal structure (FIG.25D). Residues Ser204^(5.43) and Ser207^(5.46) are critical forcatecholamine binding and activation of the β₂AR, with Ser204^(5.43)hydrogen bonding to the meta-hydroxyl and Ser207^(5.46) to thepara-hydroxyl of the catechol ring, respectively (Strader, et al., JBiol Chem 264, 13572 (1989)). In our model, the catechol hydroxyls ofisoproterenol face the appropriate serines on helix V, but the distancesare too long for hydrogen bonding (6.8 Å from meta-hydroxyl oxygen tothe sidechain oxygen of Ser204^(5.43), 4.8 Å from the para-hydroxyloxygen to the sidechain oxygen of Ser207^(5.46)). In addition,Asn293^(6.55) and Tyr308^(7.35), two residues expected to form selectiveinteractions with agonists based on the literature (Wieland, et al.,Proc Nad Acad Sci U S A 93, 9276 (1996); Kikkawa, et al., Mol Pharmacol53, 128 (1998)), are too distant to form productive polar or hydrophobiccontacts with the modeled isoproteronol molecule. These observationssuggest that agonist binding requires changes in the binding siterelative to the carazolol-bound structure, unless common structuralcomponents of agonists and inverse agonists bind in a significantlydifferent manner.

Structural Insights into β₂AR Activation

Analysis of mutations that affect β₂AR function provides insights intostructural rearrangements that are likely to occur during receptoractivation. FIG. 26A illustrates the location of amino acids for whichmutations lead to elevated basal, agonist-independent activity(constitutively active mutations, CAMs), as well as amino acids forwhich mutations impair agonist activation (uncoupling mutations, UCMs).Residues for which CAMs have been described are likely to be involved ininteractions that maintain the receptor in the inactive conformation.These amino acids are centrally located on helices III and VI. Incontrast, positions in which UCMs have been observed are likely to formintramolecular interactions that stabilize the active state. A clusterof UCMs are found at the cytoplasmic end of helix VII. Neither CAMs norUCMs are directly involved in agonist binding. Although the CAMs andUCMs are not directly connected in sequence, it is evident from thestructure that they are linked through packing interactions, such thatmovements in one will likely affect the packing of others. For example,FIG. 26A (right panel) shows all amino acids with atoms within 4 Å ofthe two centrally located CAMs, Leu124^(3.43) (Tao, et al., MolEndocrinol 14, 1272 (2000)) and Leu272^(6.34) (Jensen et al., J BiolChem 276, 9279 (2001)). Several amino acids that pack against these CAMsalso interact with one or more UCMs. Trp286^(6.48) lies at the base ofthe binding pocket. It has been proposed that agonist binding leads to achange in the rotameric state of Trp286^(6.48) with subsequent changesin the angle of the helical kink formed by Pro288^(6.86) (Shi et al., JBiol Chem 277, 40989 (2002)). It is likely that an agonist-inducedchange in the rotameric state of Trp286^(6.48) will be linked to changesin sidechains of CAMs and UCMs through packing interactions andpropagated to the cytoplasmic ends of the helices and the associatedintracellular loops that interact with G proteins and other signalingmolecules.

In the structures of both rhodopsin and the β₂AR, a cluster of watermolecules lies near the most highly conserved class A GPCR residues(FIG. 26B). It has been proposed that these water molecules may play arole in the structural changes involved in receptor activation (Pardo,et al., Chembiochem 8, 19 (2007)). FIG. 26C shows the network ofpotential hydrogen bonding interactions that link Trp286^(6.48) withconserved amino acids extending to the cytoplasmic ends of helices. UCMshave been identified for three amino acids linked by thisnetwork—N322^(7.49), P323^(7.80), and Y326^(7.83) (Barak et al.,Biochemistry 34, 15407 (1995)). This relatively loose-packed, waterfilled region is likely to be important in allowing conformationaltransitions, as there will be fewer steric restraints to sidechainrepacking.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

APPENDIX 1 HEADER MEMBRANE PROTEIN/HYDROLASE   05-OCT-07 2RH1 TITLE HIGHRESOLUTION CRYSTAL STRUCTURE OF HUMAN B2-ADRENERGIC G TITLE 2PROTEIN-COUPLED RECEPTOR COMPND   MOL_ID: 1; COMPND  2 MOLECULE:BETA-2-ADRENERGIC RECEPTOR/T4-LYSOZYME CHIMERA; COMPND  3 CHAIN: A;COMPND  4 SYNONYM: BETA-2 ADRENERGIC RECEPTOR, BETA-2 ADRENOCEPTOR,COMPND  5 BETA-2 ADRENORECEPTOR/LYSIS PROTEIN, MURAMIDASE, COMPND  6ENDOLYSIN; COMPND  7 ENGINEERED: YES; COMPND  8 MUTATION: YES SOURCE MOL_ID: 1; SOURCE  2 ORGANISM_SCIENTIFIC: HOMO SAPIENS, ENTEROBACTERIAPHAGE T4; SOURCE  3 ORGANISM_COMMON: HUMAN,; SOURCE  4 STRAIN: ,; SOURCE 5 GENE: ADRB2, ADRB2R, B2AR/E; SOURCE  6 EXPRESSION_SYSTEM: SPODOPTERAFRUGIPERDA; SOURCE  7 EXPRESSION_SYSTEM_COMMON: FALL ARMYWORM; SOURCE  8EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID; SOURCE  9EXPRESSION_SYSTEM_PLASMID: PFASTBAC1; SOURCE 10 OTHER_DETAILS: THECONSTRUCT HAS BEEN OBTAINED BY SOURCE 11 OVERLAPPING EXTENSION PCRKEYWDS  GPCR, 7TM, ADRENERGIC, FUSION, LIPIDIC CUBIC PHASE, LIPIDIC,KEYWDS 2 MESOPHASE, CHOLESTEROL, MEMBRANE PROTEIN, MEMBRANE PROTEINKEYWDS 3/HYDROLASE COMPLEX EXPDTA X-RAY DIFFRACTION AUTHOR  V. CHEREZOV,D. M. ROSENBAUM, M. A. HANSON, S. G. F. RASMUSSEN, AUTHOR 2 F. S. THIAN,T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, B. K. KOBILKA, AUTHOR 3R. C. STEVENS REVDAT 5 07-OCT-08 2RH1 1 REMARK REVDAT 4 11-DEC-07 2RH1 1REMARK REVDAT 3 04-DEC-07 2RH1 1 JRNL REVDAT 2 06-NOV-07 2RH1 1JRNL HELIX SHEET REVDAT 1 30-OCT-07 2RH1 0 JRNL AUTH V. CHEREZOV, D. M.ROSENBAUM, M. A. HANSON, S. G. RASMUSSEN, JRNL AUTH 2 F. S. THIAN, T. S.KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, JRNL AUTH 3 B. K. KOBILKA, R.C. STEVENS JRNL TITL HIGH-RESOLUTION CRYSTAL STRUCTURE OF AN ENGINEEREDJRNL TITL 2 HUMAN BETA2-ADRENERGIC G PROTEIN-COUPLED RECEPTOR. JRNLREF SCIENCE     V. 318 1258 2007 JRNL REFN ASTM SCIEAS US ISSN 0036-8075REMARK 1 REMARK 1 REFERENCE 1 REMARK 1  AUTH D. M. ROSENBAUM, V.CHEREZOV, M. A. HANSON, REMARK 1  AUTH 2 S. G. F. RASMUSSEN, F. S.THIAN, T. S. KOBILKA, H. J. CHOI, REMARK 1  AUTH 3 X. J. YAO, W. I.WEIS, R. C. STEVENS, B. K. KOBILKA REMARK 1  TITL GPCR ENGINEERINGYIELDS HIGH-RESOLUTION STRUCTURAL REMARK 1  TITL 2 INSIGHTS INTO BETA2ADRENERGIC RECEPTOR FUNCTION. REMARK 1  REF TO BE PUBLISHED REMARK 1 REFN REMARK 2 REMARK 2 RESOLUTION. 2.40 ANGSTROMS. REMARK 3 REMARK 3REFINEMENT. REMARK 3  PROGRAM: REFMAC 5.2.0019 REMARK 3 AUTHORS: MURSHUDOV, VAGIN, DODSON REMARK 3 REMARK 3  REFINEMENT TARGET:MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS): 2.40 REMARK 3  RESOLUTION RANGE LOW(ANGSTROMS): 19.95 REMARK 3  DATA CUTOFF  (SIGMA(F)): 0.000 REMARK 3 COMPLETENESS FOR RANGE  (%): 99.8 REMARK 3  NUMBER OFREFLECTIONS:   26506 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT.REMARK 3  CROSS-VALIDATION METHOD:  THROUGHOUT REMARK 3  FREE R VALUETEST SET SELECTION: RANDOM REMARK 3  R VALUE (WORKING + TEST SET): 0.198REMARK 3  R VALUE  (WORKING SET): 0.196 REMARK 3  FREE RVALUE:     0.232 REMARK 3  FREE R VALUE TEST SET SIZE (%): 4.900 REMARK3  FREE R VALUE TEST SET COUNT: 1310 REMARK 3 REMARK 3 FIT IN THEHIGHEST RESOLUTION BIN. REMARK 3  TOTAL NUMBER OF BINS USED: 20 REMARK 3 BIN RESOLUTION RANGE HIGH: 2.40 REMARK 3  BIN RESOLUTION RANGE LOW:2.46 REMARK 3  REFLECTION IN BIN (WORKING SET): 1829 REMARK 3  BINCOMPLETENESS (WORKING + TEST) (%): 98.76 REMARK 3  BIN R VALUE (WORKINGSET): 0.2700 REMARK 3  BIN FREE R VALUE SET COUNT:  78 REMARK 3  BINFREE R VALUE:    0.3010 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMSUSED IN REFINEMENT. REMARK 3  ALL ATOMS:   3804 REMARK 3 REMARK 3 BVALUES. REMARK 3  FROM WILSON PLOT  (A**2): 63.91 REMARK 3  MEAN BVALUE (OVERALL, A**2): 63.91 REMARK 3  OVERALL ANISOTROPIC B VALUE.REMARK 3  B11 (A**2): 0.43000 REMARK 3  B22 (A**2): −3.46000 REMARK 3 B33 (A**2): 4.25000 REMARK 3  B12 (A**2): 0.00000 REMARK 3  B13 (A**2):2.26000 REMARK 3  B23 (A**2): 0.00000 REMARK 3 REMARK 3 ESTIMATEDOVERALL COORDINATE ERROR. REMARK 3  ESU BASED ON R VALUE     (A): 0.295REMARK 3  ESU BASED ON FREE R VALUE    (A):0.220 REMARK 3  ESU BASED ONMAXIMUM LIKELIHOOD   (A): 0.203 REMARK 3  ESU FOR B VALUES BASED ONMAXIMUM LIKELIHOOD (A**2): 18.501 REMARK 3 REMARK 3 CORRELATIONCOEFFICIENTS. REMARK 3  CORRELATION COEFFICIENT FO-FC:  0.963 REMARK 3 CORRELATION COEFFICIENT FO-FC FREE: 0.944 REMARK 3 REMARK 3 RMSDEVIATIONS FROM IDEAL VALUES  COUNT RMS WEIGHT REMARK 3  BOND LENGTHSREFINED ATOMS  (A): 3843; 0.013; 0.022 REMARK 3  BOND LENGTHSOTHERS   (A): 2622; 0.000; 0.020 REMARK 3  BOND ANGLES REFINEDATOMS (DEGREES): 5219; 1.500; 2.000 REMARK 3  BOND ANGLESOTHERS  (DEGREES): 6377; 4.099; 3.002 REMARK 3  TORSION ANGLES, PERIOD1 (DEGREES): 441; 3.876; 5.000 REMARK 3  TORSION ANGLES, PERIOD2 (DEGREES): 154; 31.604; 23.182 REMARK 3  TORSION ANGLES, PERIOD3 (DEGREES): 627; 11.383; 15.000 REMARK 3  TORSION ANGLES, PERIOD4 (DEGREES):  22; 12.164; 15.000 REMARK 3  CHIRAL-CENTERRESTRAINTS  (A**3): 610; 0.060; 0.200 REMARK 3  GENERAL PLANES REFINEDATOMS  (A): 4008; 0.001; 0.020 REMARK 3  GENERAL PLANES OTHERS   (A):804; 0.001; 0.020 REMARK 3  NON-BONDED CONTACTS REFINED ATOMS (A): 926;0.187; 0.300 REMARK 3  NON-BONDED CONTACTS OTHERS  (A): 2443; 0.194;0.300 REMARK 3  NON-BONDED TORSION REFINED ATOMS (A): 1935; 0.180; 0.500REMARK 3  NON-BONDED TORSION OTHERS  (A): 1580; 0.107; 0.500 REMARK 3 H-BOND (X . . . Y) REFINED ATOMS  (A): 194; 0.164; 0.500 REMARK 3 H-BOND (X . . . Y) OTHERS   (A): NULL; NULL; NULL REMARK 3  POTENTIALMETAL-ION REFINED ATOMS (A): NULL; NULL; NULL REMARK 3  POTENTIALMETAL-ION OTHERS  (A): NULL; NULL; NULL REMARK 3  SYMMETRY VDW REFINEDATOMS  (A): 7; 0.155; 0.300 REMARK 3  SYMMETRY VDW OTHERS  (A): 29;0.140; 0.300 REMARK 3  SYMMETRY H-BOND REFINED ATOMS (A): 6; 0.192;0.500 REMARK 3  SYMMETRY H-BOND OTHERS  (A): NULL; NULL; NULL REMARK 3 SYMMETRY METAL-ION REFINED ATOMS (A): NULL; NULL; NULL REMARK 3 SYMMETRY METAL-ION OTHERS  (A): NULL; NULL; NULL REMARK 3 REMARK 3ISOTROPIC THERMAL FACTOR RESTRAINTS.  COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 2884; 2.352; 2.000 REMARK 3 MAIN-CHAIN BOND OTHER ATOMS (A**2): 896; 0.097; 2.000 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 3571; 2.767; 2.500 REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A**2): 1974; 4.582; 2.000 REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2): 1648; 5.849; 2.500 REMARK 3REMARK 3 ANISOTROPIC THERMAL FACTOR RESTRAINTS.  COUNT RMS WEIGHT REMARK3  RIGID-BOND RESTRAINTS  (A**2): NULL; NULL; NULL REMARK 3  SPHERICITY;FREE ATOMS  (A**2): NULL; NULL; NULL REMARK 3  SPHERICITY; BONDEDATOMS  (A**2): NULL; NULL; NULL REMARK 3 REMARK 3 NCS RESTRAINTSSTATISTICS REMARK 3  NUMBER OF DIFFERENT NCS GROUPS: NULL REMARK 3REMARK 3 TLS DETAILS REMARK 3  NUMBER OF TLS GROUPS: 3 REMARK 3 REMARK 3 TLS GROUP: 1 REMARK 3  NUMBER OF COMPONENTS GROUP: 2 REMARK 3 COMPONENTS  C SSSEQI TO C SSSEQI REMARK 3  RESIDUE RANGE: A 1002  A1011 REMARK 3  RESIDUE RANGE: A 1062  A 1161 REMARK 3  ORIGIN FOR THEGROUP (A): −23.6840 58.4050 30.4850 REMARK 3  T TENSOR REMARK 3  T11:−0.2208 T22: −0.2598 REMARK 3  T33: −0.7033 T12:  0.0432 REMARK 3  T13:−0.0594 T23: −0.0241 REMARK 3  L TENSOR REMARK 3  L11:  3.1220 L22: 2.6914 REMARK 3  L33:  5.9787 L12:  0.8643 REMARK 3  L13:  0.6146 L23:−0.8632 REMARK 3  S TENSOR REMARK 3  S11: −0.1453 S12: −0.2225 S13: 0.2116 REMARK 3  S21:  0.0984 S22: −0.0656 S23: −0.0133 REMARK 3  S31:−0.1849 S32: −0.1761 S33:  0.2109 REMARK 3 REMARK 3 TLS GROUP: 2 REMARK3  NUMBER OF COMPONENTS GROUP: 1 REMARK 3  COMPONENTS  C SSSEQI TO CSSSEQI REMARK 3  RESIDUE RANGE: A 1012  A 1061 REMARK 3  ORIGIN FOR THEGROUP (A): −35.0580 69.0010 11.9610 REMARK 3  T TENSOR REMARK 3  T11: 0.0414 T22: −0.0871 REMARK 3  T33: −0.4908 T12:  0.0577 REMARK 3  T13:−0.1559 T23: −0.0085 REMARK 3  L TENSOR REMARK 3  L11:  9.6406 L22:16.6501 REMARK 3  L33:  7.1133 L12: −6.5961 REMARK 3  L13: −0.9803 L23: 3.2882 REMARK 3  S TENSOR REMARK 3  S11: −0.1628 S12: −0.1008 S13:0.4114 REMARK 3  S21: −0.7585 S22: −0.1058 S23: 0.7355 REMARK 3  S31:−0.6590 S32: −0.6073 S33: 0.2686 REMARK 3 REMARK 3  TLS GROUP: 3 REMARK3  NUMBER OF COMPONENTS GROUP: 2 REMARK 3  COMPONENTS  C SSSEQI TO CSSSEQI REMARK 3  RESIDUE RANGE: A  29  A 230 REMARK 3  RESIDUERANGE: A 263  A 342 REMARK 3  ORIGIN FOR THE GROUP (A): −33.074020.0130 7.1220 REMARK 3  T TENSOR REMARK 3  T11: −0.0103 T22: −0.2341REMARK 3  T33: −0.5401 T12: −0.0025 REMARK 3  T13: −0.0974 T23: −0.0034REMARK 3  L TENSOR REMARK 3  L11:  2.3670 L22: 6.1551 REMARK 3  L33: 1.9314 L12: 2.1068 REMARK 3  L13:  0.8591 L23: 0.7864 REMARK 3  STENSOR REMARK 3  S11: −0.0346 S12:  0.0267 S13: −0.2068 REMARK 3  S21:−0.5009 S22:  0.0712 S23:  0.2388 REMARK 3  S31:  0.3208 S32:  0.0002S33: −0.0366 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3  METHODUSED: MASK REMARK 3  PARAMETERS FOR MASK CALCULATION REMARK 3  VDW PROBERADIUS: 1.40 REMARK 3  ION PROBE RADIUS: 0.80 REMARK 3  SHRINKAGERADIUS: 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: HYDROGENS HAVEBEEN ADDED IN THE REMARK 3 RIDING POSITIONS. WATER #548 HAS STRONGDIFFERENCE DENSITY BUT REMARK 3 WEAK 2FO-FC DENSITY. REMARK 4 REMARK 42RH1 COMPLIES WITH FORMAT V. 3.1, 01-AUG-2007 REMARK 100 REMARK 100 THISENTRY HAS BEEN PROCESSED BY RCSB. REMARK 100 THE RCSB ID CODE ISRCSB044849. REMARK 200 REMARK 200 EXPERIMENTAL DETAILS REMARK 200 EXPERIMENT TYPE:   X-RAY DIFFRACTION REMARK 200  DATE OF DATACOLLECTION:  22-JUN-2007; 18-JUL-2007 REMARK 200 TEMPERATURE   (KELVIN): 78; 78 REMARK 200  PH:     6.75 REMARK 200 NUMBER OF CRYSTALS USED:  27 REMARK 200 REMARK 200 SYNCHROTRON   (Y/N): Y; Y REMARK 200  RADIATION SOURCE:   APS; APSREMARK 200  BEAMLINE:    23-ID-B; 23-ID-B REMARK 200  X-RAY GENERATORMODEL:  NULL REMARK 200  MONOCHROMATIC OR LAUE (M/L): M REMARK 200 WAVELENGTH OR RANGE  (A):1.03321; 1.03321 REMARK 200MONOCHROMATOR:   DOUBLE CRYSTAL REMARK 200 OPTICS:     MIRRORS; MIRRORSREMARK 200 REMARK 200 DETECTOR TYPE:   CCD; CCD REMARK 200 DETECTORMANUFACTURER:  MARMOSAIC 300 MM CCD; REMARK 200       MARMOSAIC 300 MMCCD REMARK 200 INTENSITY-INTEGRATION SOFTWARE: XDS REMARK 200 DATASCALING SOFTWARE:  XDS REMARK 200 REMARK 200 NUMBER OF UNIQUEREFLECTIONS: 26506 REMARK 200 RESOLUTION RANGE HIGH (A): 2.400 REMARK200 RESOLUTION RANGE LOW (A): 20.000 REMARK 200 REJECTION CRITERIA(SIGMA(I)): −3.000 REMARK 200 REMARK 200 OVERALL. REMARK 200 COMPLETENESS FOR RANGE (%): 99.2 REMARK 200  DATA REDUNDANCY:   NULLREMARK 200  R MERGE    (I): 0.12700 REMARK 200  R SYM    (I): NULLREMARK 200  <I/SIGMA(I)> FOR THE DATA SET: 9.6200 REMARK 200 REMARK 200IN THE HIGHEST RESOLUTION SHELL. REMARK 200  HIGHEST RESOLUTION SHELL,RANGE HIGH (A): 2.40 REMARK 200  HIGHEST RESOLUTION SHELL, RANGE LOW(A): 2.50 REMARK 200  COMPLETENESS FOR SHELL  (%): 99.1 REMARK 200  DATAREDUNDANCY IN SHELL:  NULL REMARK 200  R MERGE FOR SHELL   (I): 0.67800REMARK 200  R SYM FOR SHELL   (I): NULL REMARK 200  <I/SIGMA(I)> FORSHELL:   2.200 REMARK 200 REMARK 200 DIFFRACTION PROTOCOL: SINGLEWAVELENGTH REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULARREPLACEMENT REMARK 200 SOFTWARE USED: PHASER REMARK 200 STARTING MODEL:PDB ENTRIES 1U19, 2LZM REMARK 200 REMARK 200 REMARK: THIS STRUCTURE IS APART OF THE ROADMAP/PSI COMMUNITY REMARK 200  OUTREACH PROGRAM, NOT ASPECIFIC PSI TARGET. REMARK 280 REMARK 280 CRYSTAL REMARK 280 SOLVENTCONTENT, VS (%): 59.98 REMARK 280 MATTHEWS COEFFICIENT, VM(ANGSTROMS**3/DA): 3.07 REMARK 280 REMARK 280 CRYSTALLIZATIONCONDITIONS: 30-35% V/V PEG 400, 0.1-0.2 M REMARK 280  NA2SO4, 0.1 MBIS-TRIS PROPANE PH 6.5-7.0, 5-7% 1,4- REMARK 280  BUTANEDIOL, 8-10%CHOLESTEROL, 52-50% MONOOLEIN, PH 6.75, REMARK 280  LIPIDIC MESOPHASE,TEMPERATURE 293 K REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRY REMARK290 SYMMETRY OPERATORS FOR SPACE GROUP: C 1 2 1 REMARK 290 REMARK 290  SYMOP SYMMETRY REMARK 290  NNNMMM OPERATOR REMARK 290   1555 X, Y, ZREMARK 290   2555 −X, Y, −Z REMARK 290   3555 ½ + X, ½ + Y, Z REMARK 290  4555 ½ − X, ½ + Y, −Z REMARK 290 REMARK 290  WHERE NNN -> OPERATORNUMBER REMARK 290    MMM -> TRANSLATION VECTOR REMARK 290CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS REMARK 290 THE FOLLOWINGTRANSFORMATIONS OPERATE ON THE ATOM/HETATM REMARK 290 RECORDS IN THISENTRY TO PRODUCE CRYSTALLOGRAPHICALLY REMARK 290 RELATED MOLECULES.REMARK 290  SMTRY1 1 1.000000 0.000000  0.000000  0.00000 REMARK 290 SMTRY2 1 0.000000 1.000000  0.000000  0.00000 REMARK 290  SMTRY3 10.000000 0.000000  1.000000  0.00000 REMARK 290  SMTRY1 2 −1.0000000.000000  0.000000  0.00000 REMARK 290  SMTRY2 2 0.000000 1.000000 0.000000  0.00000 REMARK 290  SMTRY3 2 0.000000 0.000000 −1.000000 0.00000 REMARK 290  SMTRY1 3 1.000000 0.000000  0.000000 53.15900REMARK 290  SMTRY2 3 0.000000 1.000000  0.000000 84.62000 REMARK 290 SMTRY3 3 0.000000 0.000000  1.000000  0.00000 REMARK 290  SMTRY1 4−1.000000 0.000000  0.000000 53.15900 REMARK 290  SMTRY2 4 0.0000001.000000  0.000000 84.62000 REMARK 290  SMTRY3 4 0.000000 0.000000−1.000000  0.00000 REMARK 290 REMARK 290 REMARK: NULL REMARK 300 REMARK300 BIOMOLECULE: 1 REMARK 300 SEE REMARK 350 FOR THE AUTHOR PROVIDEDAND/OR PROGRAM REMARK 300 GENERATED ASSEMBLY INFORMATION FOR THESTRUCTURE IN REMARK 300 THIS ENTRY. THE REMARK MAY ALSO PROVIDEINFORMATION ON REMARK 300 BURIED SURFACE AREA. REMARK 300 REMARK 300REMARK: AUTHORS STATE THAT THE BIOLOGICAL UNIT IS UNKNOWN REMARK 350REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWNREMARK 350 BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE REMARK350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS REMARK350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND REMARK 350CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN. REMARK 350 REMARK 350BIOMOLECULE: 1 REMARK 350 SOFTWARE DETERMINED QUATERNARY STRUCTURE:MONOMERIC REMARK 350 SOFTWARE USED: PISA REMARK 350 APPLY THE FOLLOWINGTO CHAINS: A REMARK 350  BIOMT1 1 1.000000 0.000000 0.000000 0.00000REMARK 350  BIOMT2 1 0.000000 1.000000 0.000000 0.00000 REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000 REMARK 375 REMARK 375SPECIAL POSITION REMARK 375 THE FOLLOWING ATOMS ARE FOUND TO BE WITHIN0.15 ANGSTROMS REMARK 375 OF A SYMMETRY RELATED ATOM AND ARE ASSUMED TOBE ON SPECIAL REMARK 375 POSITIONS. REMARK 375 REMARK 375 ATOM RESCSSEQI REMARK 375  HOH A 520 LIES ON A SPECIAL POSITION. REMARK 465REMARK 465 MISSING RESIDUES REMARK 465 THE FOLLOWING RESIDUES WERE NOTLOCATED IN THE REMARK 465 EXPERIMENT. (M = MODEL NUMBER; RES = RESIDUENAME; C = CHAIN REMARK 465 IDENTIFIER; SSEQ = SEQUENCE NUMBER; I =INSERTION CODE.) REMARK 465 REMARK 465  M RES C SSEQI REMARK 465  ASPA −6 REMARK 465  TYR A −5 REMARK 465  LYS A −4 REMARK 465  ASP A −3REMARK 465  ASP A −2 REMARK 465  ASP A −1 REMARK 465  ALA A 0 REMARK 465 MET A 1 REMARK 465  GLY A 2 REMARK 465  GLN A 3 REMARK 465  PRO A 4REMARK 465  GLY A 5 REMARK 465  ASN A 6 REMARK 465  GLY A 7 REMARK 465 SER A 8 REMARK 465  ALA A 9 REMARK 465  PHE A 10 REMARK 465  LEU A 11REMARK 465  LEU A 12 REMARK 465  ALA A 13 REMARK 465  PRO A 14 REMARK465  ASN A 15 REMARK 465  ARG A 16 REMARK 465  SER A 17 REMARK 465  HISA 18 REMARK 465  ALA A 19 REMARK 465  PRO A 20 REMARK 465  ASP A 21REMARK 465  HIS A 22 REMARK 465  ASP A 23 REMARK 465  VAL A 24 REMARK465  THR A 25 REMARK 465  GLN A 26 REMARK 465  GLN A 27 REMARK 465  ARGA 28 REMARK 465  ARG A 343 REMARK 465  ARG A 344 REMARK 465  SER A 345REMARK 465  SER A 346 REMARK 465  LEU A 347 REMARK 465  LYS A 348 REMARK465  ALA A 349 REMARK 465  TYR A 350 REMARK 465  GLY A 351 REMARK 465 ASN A 352 REMARK 465  GLY A 353 REMARK 465  TYR A 354 REMARK 465  SERA 355 REMARK 465  SER A 356 REMARK 465  ASN A 357 REMARK 465  GLY A 358REMARK 465  ASN A 359 REMARK 465  THR A 360 REMARK 465  GLY A 361 REMARK465  GLU A 362 REMARK 465  GLN A 363 REMARK 465  SER A 364 REMARK 465 GLY A 365 REMARK 470 REMARK 470 MISSING ATOM REMARK 470 THE FOLLOWINGRESIDUES HAVE MISSING ATOMS(M = MODEL NUMBER; REMARK 470 RES = RESIDUENAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER; REMARK 470 I =INSERTION CODE): REMARK 470  M RES CSSEQI ATOMS REMARK 470  ASP A29 CG OD1 OD2 REMARK 500 REMARK 500 GEOMETRY AND STEREOCHEMISTRY REMARK500 SUBTOPIC: CLOSE CONTACTS IN SAME ASYMMETRIC UNIT REMARK 500 REMARK500 THE FOLLOWING ATOMS ARE IN CLOSE CONTACT. REMARK 500 REMARK 500 ATM1RES C SSEQI ATM2 RES C SSEQI REMARK 500  SG1 CYS A  341 O2 PLMA 415   1.88 REMARK 500 REMARK 500 REMARK: NULL REMARK 500 REMARK 500GEOMETRY AND STEREOCHEMISTRY REMARK 500 SUBTOPIC: TORSION ANGLES REMARK500 REMARK 500 TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS:REMARK 500 (M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN IDENTIFIER;REMARK 500 SSEQ = SEQUENCE NUMBER; I = INSERTION CODE). REMARK 500REMARK 500 STANDARD TABLE: REMARK 500 FORMAT: (10X, I3, 1X, A3, 1X, A1,I4, A1, 4X, F7.2, 3X, F7.2) REMARK 500 REMARK 500 EXPECTED VALUES: GJKLEYWEGT AND TA JONES (1996). PHI/PSI- REMARK 500 CHOLOGY: RAMACHANDRANREVISITED. STRUCTURE 4, 1395-1400 REMARK 500 REMARK 500 M RESCSSEQI  PSI  PHI REMARK 500  VAL A 86  −64.27 −100.05 REMARK 500  TYR A141  −22.19  73.12 REMARK 500 REMARK 500 REMARK: NULL REMARK 600 REMARK600 HETEROGEN REMARK 600 THE PALMITIC ACID (PLM) AND ACETAMIDE (ACM)GROUPS ARE REMARK 600 COVALENTLY LINKED TO THEIR RESPECTIVE CYSTEINERESIDUES. REMARK 610 REMARK 610 MISSING HETEROATOM REMARK 610 THEFOLLOWING RESIDUES HAVE MISSING ATOMS (M = MODEL NUMBER; REMARK 610 RES= RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER; REMARK 610I = INSERTION CODE): REMARK 610  M RES C SSEQI REMARK 610  12P A 416REMARK 800 REMARK 800 SITE REMARK 800 SITE_IDENTIFIER: AC1 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE 12P A 416 REMARK 800SITE_IDENTIFIER: AC2 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE ACM A 411 REMARK 800 SITE_IDENTIFIER: AC3 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE BU1 A 409 REMARK 800SITE_IDENTIFIER: AC5 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE CAU A 408 REMARK 800 SITE_IDENTIFIER: AC6 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CLR A 412 REMARK 800SITE_IDENTIFIER: AC7 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE CLR A 413 REMARK 800 SITE_IDENTIFIER: AC9 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE MAL A 401 REMARK 800SITE_IDENTIFIER: BC1 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE PLM A 415 REMARK 800 SITE_IDENTIFIER: BC2 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 402 REMARK 800SITE_IDENTIFIER: BC3 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE SO4 A 403 REMARK 800 SITE_IDENTIFIER: BC4 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 404 REMARK 800SITE_IDENTIFIER: BC5 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE SO4 A 405 REMARK 800 SITE_IDENTIFIER: BC6 REMARK 800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 406 REMARK 800SITE_IDENTIFIER: BC7 REMARK 800 SITE_DESCRIPTION: BINDING SITE FORRESIDUE SO4 A 407 REMARK 999 REMARK 999 SEQUENCE THE STRUCTURE IS ANINTERNAL FUSION PROTEIN WITH REMARK 999 LYSOZYME. AN OFFSET 1000 HASBEEN ADDED TO ORIGINAL REMARK 999 SEQUENCE DATABASE RESIDUE NUMBERS(2-161) OF THE LYSOZYME REMARK 999 PART IN COORDINATES TO DISTINGUISHTHE LYSOZYME PART IN THE REMARK 999 CHAIN. THEREFORE THE RESIDUES OFLYSOZYME PART HAVE NUMBERS REMARK 999 A1002-A1161. DBREF 2RH1 A 1 230 UNP P07550 ADRB2_HUMAN  1 230 DBREF 2RH1 A1002 1161 UNP  P00720 LYS_BPT4  2  161 DBREF 2RH1 A 263 365 UNP  P07550 ADRB2_HUMAN 263  365 SEQADV 2RH1 ASP A  −6 UNPP07550 EXPRESSION TAG SEQADV 2RH1 TYR A  −5 UNP P07550 EXPRESSION TAGSEQADV 2RH1 LYS A  −4 UNP P07550 EXPRESSION TAG SEQADV 2RH1 ASP A  −3UNP P07550 EXPRESSION TAG SEQADV 2RH1 ASP A  −2 UNP P07550 EXPRESSIONTAG SEQADV 2RH1 ASP A  −1 UNP P07550 EXPRESSION TAG SEQADV 2RH1 ALA A  0UNP P07550 EXPRESSION TAG SEQADV 2RH1 GLU A  187 UNP P07550 ASN 187ENGINEERED SEQADV 2RH1 THR A  1054 UNP P00720 CYS 54 ENGINEERED SEQADV2RH1 ALA A 1097 UNP P00720 CYS 97 ENGINEERED SEQRES 1 A 500 ASP TYR LYSASP ASP ASP ALA MET GLY GLN PRO GLY ASN SEQRES 2 A 500 GLY SER ALA PHELEU LEU ALA PRO ASN ARG SER HIS ALA SEQRES 3 A 500 PRO ASP HIS ASP VALTHR GLN GLN ARG ASP GLU VAL TRP SEQRES 4 A 500 VAL VAL GLY MET GLY ILEVAL MET SER LEU ILE VAL LEU SEQRES 5 A 500 ALA ILE VAL PHE GLY ASN VALLEU VAL ILE THR ALA ILE SEQRES 6 A 500 ALA LYS PHE GLU ARG LEU GLN THRVAL THR ASN TYR PHE SEQRES 7 A 500 ILE THR SER LEU ALA CYS ALA ASP LEUVAL MET GLY LEU SEQRES 8 A 500 ALA VAL VAL PRO PHE GLY ALA ALA HIS ILELEU MET LYS SEQRES 9 A 500 MET TRP THR PHE GLY ASN PHE TRP CYS GLU PHETRP THR SEQRES 10 A 500 SER ILE ASP VAL LEU CYS VAL THR ALA SER ILE GLUTHR SEQRES 11 A 500 LEU CYS VAL ILE ALA VAL ASP ARG TYR PHE ALA ILE THRSEQRES 12 A 500 SER PRO PHE LYS TYR GLN SER LEU LEU THR LYS ASN LYSSEQRES 13 A 500 ALA ARG VAL ILE ILE LEU MET VAL TRP ILE VAL SER GLYSEQRES 14 A 500 LEU THR SER PHE LEU PRO ILE GLN MET HIS TRP TYR ARGSEQRES 15 A 500 ALA THR HIS GLN GLU ALA ILE ASN CYS TYR ALA GLU GLUSEQRES 16 A 500 THR CYS CYS ASP PHE PHE THR ASN GLN ALA TYR ALA ILESEQRES 17 A 500 ALA SER SER ILE VAL SER PHE TYR VAL PRO LEU VAL ILESEQRES 18 A 500 MET VAL PHE VAL TYR SER ARG VAL PHE GLN GLU ALA LYSSEQRES 19 A 500 ARG GLN LEU ASN ILE PHE GLU MET LEU ARG ILE ASP GLUSEQRES 20 A 500 GLY LEU ARG LEU LYS ILE TYR LYS ASP THR GLU GLY TYRSEQRES 21 A 500 TYR THR ILE GLY ILE GLY HIS LEU LEU THR LYS SER PROSEQRES 22 A 500 SER LEU ASN ALA ALA LYS SER GLU LEU ASP LYS ALA ILESEQRES 23 A 500 GLY ARG ASN THR ASN GLY VAL ILE THR LYS ASP GLU ALASEQRES 24 A 500 GLU LYS LEU PHE ASN GLN ASP VAL ASP ALA ALA VAL ARGSEQRES 25 A 500 GLY ILE LEU ARG ASN ALA LYS LEU LYS PRO VAL TYR ASPSEQRES 26 A 500 SER LEU ASP ALA VAL ARG ARG ALA ALA LEU ILE ASN METSEQRES 27 A 500 VAL PHE GLN MET GLY GLU THR GLY VAL ALA GLY PHE THRSEQRES 28 A 500 ASN SER LEU ARG MET LEU GLN GLN LYS ARG TRP ASP GLUSEQRES 29 A 500 ALA ALA VAL ASN LEU ALA LYS SER ARG TRP TYR ASN GLNSEQRES 30 A 500 THR PRO ASN ARG ALA LYS ARG VAL ILE THR THR PHE ARGSEQRES 31 A 500 THR GLY THR TRP ASP ALA TYR LYS PHE CYS LEU LYS GLUSEQRES 32 A 500 HIS LYS ALA LEU LYS THR LEU GLY ILE ILE MET GLY THRSEQRES 33 A 500 PHE THR LEU CYS TRP LEU PRO PHE PHE ILE VAL ASN ILESEQRES 34 A 500 VAL HIS VAL ILE GLN ASP ASN LEU ILE ARG LYS GLU VALSEQRES 35 A 500 TYR ILE LEU LEU ASN TRP ILE GLY TYR VAL ASN SER GLYSEQRES 36 A 500 PHE ASN PRO LEU ILE TYR CYS ARG SER PRO ASP PHE ARGSEQRES 37 A 500 ILE ALA PHE GLN GLU LEU LEU CYS LEU ARG ARG SER SERSEQRES 38 A 500 LEU LYS ALA TYR GLY ASN GLY TYR SER SER ASN GLY ASNSEQRES 39 A 500 THR GLY GLU GLN SER GLY HET MAL A 401  23 HET SO4 A 402 5 HET SO4 A 403  5 HET SO4 A 404  5 HET SO4 A 405  5 HET SO4 A 406  5HET SO4 A 407  5 HET CAU A 408  22 HET BU1 A 409  6 HET BU1 A 410  6 HETACM A 411  4 HET CLR A 412  28 HET CLR A 413  28 HET CLR A 414  28 HETPLM A 415  17 HET 12P A 416  21 HETNAM   MAL MALTOSE HETNAM   SO4SULFATE ION HETNAM   CAU(2S)-1-(9H-CARBAZOL-4-YLOXY)-3-(ISOPROPYLAMINO)PROPAN- HETNAM 2 CAU 2-OLHETNAM   BU1 1,4-BUTANEDIOL HETNAM   ACM ACETAMIDE HETNAM   CLRCHOLESTEROL HETNAM   PLM PALMITIC ACID HETNAM   12P DODECAETHYLENEGLYCOL HETSYN   CAU (S)-CARAZOLOL HETSYN   12P POLYETHYLENE GLYCOLPEG400 FORMUL  2 MAL  C12 H22 O11 FORMUL  3 SO4  6(O4 S 2−) FORMUL  9CAU  C18 H22 N2 O2 FORMUL 10 BU1  2(C4 H10 O2) FORMUL 12 ACM  C2 H5 N OFORMUL 13 CLR  3(C27 H46 O) FORMUL 16 PLM  C16 H32 O2 FORMUL 17 12P  C24H50 O13 FORMUL 18 HOH  *48(H2 O) HELIX  1 1 ASP A  29 LYS A  60  1 32HELIX  2 2 VAL A  67 MET A  96  1 30 HELIX  3 3 ASN A  103 THR A  136  1  34 HELIX  4 4 LYS A  147 MET A  171  1  25 HELIX  5 5 HIS A  178 GLU A 187  1  10 HELIX  6 6 GLN A  197 GLN A  229  1   33 HELIX  7 7 LYS A 267 ILE A  298  1 32 HELIX  8 8 LYS A  305 ARG A  328  1   24 HELIX  99 PRO A  330 CYS A  341  1   12 HELIX 10 10 ILE A  1003 GLU A  1011  1  9 HELIX 11 11 LEU A  1039 ILE A  1050  1   12 HELIX 12 12 LYS A  1060ARG A  1080  1    21 HELIX 13 13 ALA A  1082 SER A  1090  1     9 HELIX14 14 ALA A  1093 MET A  1106  1     14 HELIX 15 15 GLU A  1108 GLY A 1113  1     6 HELIX 16 16 THR A  1115 GLN A  1123  1     9 HELIX 17 17TRP A  1126 ALA A  1134  1     9 HELIX 18 18 ARG A  1137 GLN A  1141  1    5 HELIX 19 19 PRO A  1143 THR A  1155  1     13 SHEET  1 1 4 LYS A1016 ASP A  1020  0 SHEET  2 1 4 TYR A 1024 GLY A  1028  0 SHEET  3 1 4HIS A 1031 THR A  1034  0 SHEET  4 1 4 GLY A 105 THR A  1059  0 SSBOND 1CYS A 106  CYS A  191 1555 1555 2.05 SSBOND 2 CYS A 184  CYS A  190 15551555 2.06 LINK SG CYS A 265 C2 ACM A 411 1555 1555 1.61 LINK SG CYS A341 C1 PLM A 415 1555 1555 1.62 SITE  1 AC1 2 ASP A 1072 HOH A 538 SITE 1 AC2 1 CYS A 265 SITE  1 AC3 4 LYS A 263 PHE A 264 HIS A 269 HOH A 502SITE  1 AC5 4 ASP A 113 PHE A 193 ASN A 312 TYR A 316 SITE  1 AC6 1 ILEA 112 SITE  1 AC7 1 HOH A 520 SITE  1 AC9 7 GLU A 1011 GLY A 1030 LEU A1032 ASP A 1070 SITE  2 AC9 7 VAL A 1103 PHE A 1104 ARG A1145 SITE  1BC1 2 LEU A 339 CYS A 341 SITE  1 BC2 5 VAL A 67 THR A 68 ARG A 131 TYRA 141 SITE  2 BC2 5 SER A 143 SITE  1 BC3 4 PHE A 264 LYS A 270 LYS A273 ARG A 328 SITE  1 BC4 5 PHE A 1114 THR A 1115 ASN A 1116 SER A 1117SITE  2 BC4 5 ASN A 1132 SITE  1 BC5 6 PRO A 1143 ASN A 1144 ARG A 1145HOH A 512 SITE  2 BC5 6 HOH A 526 HOH A 531 SITE  1 BC6 1 ARG A 1095SITE  1 BC7 2 LEU A 1015 LYS A 1016 CRYST1 106.318 169.240  40.154 90.00105.62 90.00 C 1 2 1   4 ORIGX1 1.000000 0.000000 0.000000 0.00000ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.0000001.000000 0.00000 SCALE1 0.009406 0.000000 0.002630 0.00000 SCALE20.000000 0.005909 0.000000 0.00000 SCALE3 0.000000 0.000000 0.0258590.00000 ATOM 1 N ASP A 29 −52.822 −1.611 23.137 1.00 98.48 N ATOM 2 CAASP A 29 −51.922 −2.262 22.148 1.00 98.06 C ATOM 3 C ASP A 29 −52.178−1.713 20.742 1.00 97.74 C ATOM 4 O ASP A 29 −51.291 −1.100 20.143 1.0096.54 O ATOM 5 CB ASP A 29 −52.106 −3.786 22.184 1.00 97.64 C ATOM 6 NGLU A 30 −53.394 −1.944 20.236 1.00 98.37 N ATOM 7 CA GLU A 30 −53.821−1.515 18.887 1.00 98.17 C ATOM 8 C GLU A 30 −54.424 −0.104 18.879 1.0098.57 C ATOM 9 O GLU A 30 −54.197 0.649 17.943 1.00 99.79 O ATOM 10 CBGLU A 30 −54.840 −2.498 18.305 1.00 99.00 C ATOM 11 CG GLU A 30 −54.377−3.969 18.286 1.00 99.46 C ATOM 12 CD GLU A 30 −55.432 −4.928 17.7331.00 98.77 C ATOM 13 OE1 GLU A 30 −56.228 −4.527 16.853 1.00 97.60 OATOM 14 OE2 GLU A 30 −55.463 −6.092 18.185 1.00 99.75 O ATOM 15 N VAL A31 −55.190 0.248 19.918 1.00 97.89 N ATOM 16 CA VAL A 31 −55.757 1.61820.079 1.00 96.48 C ATOM 17 C VAL A 31 −54.643 2.678 20.185 1.00 95.12 CATOM 18 O VAL A 31 −54.838 3.837 19.803 1.00 93.37 O ATOM 19 CB VAL A 31−56.729 1.697 21.306 1.00 96.71 C ATOM 20 CG1 VAL A 31 −57.185 3.13521.580 1.00 96.80 C ATOM 21 CG2 VAL A 31 −57.948 0.806 21.077 1.00 96.80C ATOM 22 N TRP A 32 −53.486 2.259 20.709 1.00 95.21 N ATOM 23 CA TRP A32 −52.267 3.063 20.735 1.00 94.60 C ATOM 24 C TRP A 32 −51.837 3.49119.319 1.00 93.60 C ATOM 25 O TRP A 32 −51.335 4.603 19.140 1.00 91.48 OATOM 26 CB TRP A 32 −51.129 2.273 21.427 1.00 96.93 C ATOM 27 CG TRP A32 −49.770 2.899 21.304 1.00 98.27 C ATOM 28 CD1 TRP A 32 −49.169 3.73422.197 1.00 100.48 C ATOM 29 CD2 TRP A 32 −48.844 2.737 20.216 1.00100.89 C ATOM 30 NE1 TRP A 32 −47.924 4.105 21.737 1.00 101.70 N ATOM 31CE2 TRP A 32 −47.700 3.507 20.523 1.00 102.25 C ATOM 32 CE3 TRP A 32−48.874 2.013 19.010 1.00 100.84 C ATOM 33 CZ2 TRP A 32 −46.587 3.57819.667 1.00 101.34 C ATOM 34 CZ3 TRP A 32 −47.771 2.082 18.158 1.00100.68 C ATOM 35 CH2 TRP A 32 −46.640 2.860 18.493 1.00 101.20 C ATOM 36N VAL A 33 −52.036 2.600 18.332 1.00 92.39 N ATOM 37 CA VAL A 33 −51.6672.852 16.922 1.00 91.10 C ATOM 38 C VAL A 33 −52.435 4.017 16.332 1.0089.89 C ATOM 39 O VAL A 33 −51.827 4.918 15.748 1.00 88.01 O ATOM 40 CBVAL A 33 −51.920 1.621 16.012 1.00 91.28 C ATOM 41 CG1 VAL A 33 −51.6251.950 14.553 1.00 93.68 C ATOM 42 CG2 VAL A 33 −51.080 0.443 16.467 1.0093.38 C ATOM 43 N VAL A 34 −53.762 3.986 16.490 1.00 88.20 N ATOM 44 CAVAL A 34 −54.638 5.078 16.041 1.00 87.62 C ATOM 45 C VAL A 34 −54.1456.388 16.649 1.00 87.34 C ATOM 46 O VAL A 34 −53.871 7.334 15.921 1.0088.42 O ATOM 47 CB VAL A 34 −56.126 4.852 16.422 1.00 87.61 C ATOM 48CG1 VAL A 34 −56.972 6.064 16.046 1.00 87.66 C ATOM 49 CG2 VAL A 34−56.684 3.592 15.755 1.00 85.12 C ATOM 50 N GLY A 35 −54.036 6.41317.981 1.00 86.61 N ATOM 51 CA GLY A 35 −53.524 7.576 18.738 1.00 86.08C ATOM 52 C GLY A 35 −52.123 8.048 18.358 1.00 85.30 C ATOM 53 O GLY A35 −51.837 9.245 18.408 1.00 85.51 O ATOM 54 N MET A 36 −51.257 7.11017.981 1.00 85.55 N ATOM 55 CA MET A 36 −49.919 7.431 17.486 1.00 84.86C ATOM 56 C MET A 36 −50.004 7.888 16.029 1.00 85.06 C ATOM 57 O MET A36 −49.216 8.733 15.590 1.00 86.04 O ATOM 58 CB MET A 36 −49.001 6.21417.596 1.00 86.69 C ATOM 59 CG MET A 36 −47.509 6.496 17.400 1.00 88.99C ATOM 60 SD MET A 36 −46.800 7.642 18.613 1.00 102.42 S ATOM 61 CE META 36 −47.213 6.857 20.179 1.00 100.39 C ATOM 62 N GLY A 37 −50.959 7.32415.287 1.00 83.06 N ATOM 63 CA GLY A 37 −51.227 7.713 13.911 1.00 82.58C ATOM 64 C GLY A 37 −51.710 9.144 13.811 1.00 81.74 C ATOM 65 O GLY A37 −51.171 9.912 13.024 1.00 83.08 O ATOM 66 N ILE A 38 −52.724 9.49414.611 1.00 81.34 N ATOM 67 CA ILE A 38 −53.260 10.869 14.659 1.00 80.88C ATOM 68 C ILE A 38 −52.153 11.874 15.003 1.00 80.13 C ATOM 69 O ILE A38 −52.121 12.964 14.453 1.00 81.84 O ATOM 70 CB ILE A 38 −54.432 11.03215.686 1.00 81.07 C ATOM 71 CG1 ILE A 38 −55.635 10.132 15.356 1.0081.29 C ATOM 72 CG2 ILE A 38 −54.912 12.479 15.745 1.00 80.45 C ATOM 73CD1 ILE A 38 −56.310 10.413 14.026 1.00 85.05 C ATOM 74 N VAL A 39−51.255 11.496 15.911 1.00 79.45 N ATOM 75 CA VAL A 39 −50.128 12.34616.306 1.00 79.67 C ATOM 76 C VAL A 39 −49.166 12.575 15.154 1.00 79.83C ATOM 77 O VAL A 39 −48.803 13.717 14.863 1.00 81.09 O ATOM 78 CB VAL A39 −49.352 11.740 17.496 1.00 81.11 C ATOM 79 CG1 VAL A 39 −47.99612.425 17.671 1.00 78.01 C ATOM 80 CG2 VAL A 39 −50.185 11.843 18.7701.00 81.49 C ATOM 81 N MET A 40 −48.754 11.492 14.506 1.00 80.50 N ATOM82 CA MET A 40 −47.881 11.589 13.320 1.00 80.67 C ATOM 83 C MET A 40−48.575 12.333 12.155 1.00 80.62 C ATOM 84 O MET A 40 −47.915 13.03311.390 1.00 78.80 O ATOM 85 CB MET A 40 −47.405 10.197 12.870 1.00 80.79C ATOM 86 CG MET A 40 −46.294 9.613 13.745 1.00 80.56 C ATOM 87 SD MET A40 −45.995 7.863 13.419 1.00 83.92 S ATOM 88 CE MET A 40 −44.486 7.58814.344 1.00 83.67 C ATOM 89 N SER A 41 −49.896 12.177 12.034 1.00 79.41N ATOM 90 CA SER A 41 −50.672 12.898 11.020 1.00 79.83 C ATOM 91 C SER A41 −50.651 14.403 11.269 1.00 80.21 C ATOM 92 O SER A 41 −50.521 15.18110.324 1.00 82.42 O ATOM 93 CB SER A 41 −52.104 12.394 10.976 1.00 79.47C ATOM 94 OG SER A 41 −52.128 11.044 10.561 1.00 82.68 O ATOM 95 N LEU A42 −50.778 14.805 12.534 1.00 79.85 N ATOM 96 CA LEU A 42 −50.675 16.21912.908 1.00 79.83 C ATOM 97 C LEU A 42 −49.256 16.752 12.687 1.00 78.29C ATOM 98 O LEU A 42 −49.093 17.917 12.355 1.00 80.15 O ATOM 99 CB LEU A42 −51.106 16.448 14.364 1.00 80.71 C ATOM 100 CG LEU A 42 −52.58616.242 14.714 1.00 81.76 C ATOM 101 CD1 LEU A 42 −52.774 16.400 16.2131.00 83.86 C ATOM 102 CD2 LEU A 42 −53.508 17.191 13.956 1.00 82.80 CATOM 103 N ILE A 43 −48.243 15.902 12.876 1.00 77.38 N ATOM 104 CA ILE A43 −46.850 16.274 12.593 1.00 77.38 C ATOM 105 C ILE A 43 −46.683 16.60411.112 1.00 77.46 C ATOM 106 O ILE A 43 −46.072 17.608 10.771 1.00 77.58O ATOM 107 CB ILE A 43 −45.845 15.155 12.995 1.00 78.54 C ATOM 108 CG1ILE A 43 −45.698 15.080 14.520 1.00 80.06 C ATOM 109 CG2 ILE A 43−44.473 15.387 12.366 1.00 74.64 C ATOM 110 CD1 ILE A 43 −44.923 13.84515.002 1.00 77.13 C ATOM 111 N VAL A 44 −47.229 15.750 10.247 1.00 77.58N ATOM 112 CA VAL A 44 −47.195 15.967 8.801 1.00 76.60 C ATOM 113 C VALA 44 −47.951 17.251 8.416 1.00 78.14 C ATOM 114 O VAL A 44 −47.44518.053 7.629 1.00 76.84 O ATOM 115 CB VAL A 44 −47.769 14.755 8.041 1.0076.85 C ATOM 116 CG1 VAL A 44 −47.967 15.079 6.567 1.00 75.75 C ATOM 117CG2 VAL A 44 −46.856 13.533 8.219 1.00 70.47 C ATOM 118 N LEU A 45−49.150 17.434 8.972 1.00 78.66 N ATOM 119 CA LEU A 45 −49.954 18.6388.719 1.00 78.89 C ATOM 120 C LEU A 45 −49.219 19.901 9.163 1.00 79.68 CATOM 121 O LEU A 45 −49.246 20.900 8.460 1.00 80.46 O ATOM 122 CB LEU A45 −51.306 18.555 9.433 1.00 80.42 C ATOM 123 CG LEU A 45 −52.374 19.5909.062 1.00 83.12 C ATOM 124 CD1 LEU A 45 −52.835 19.414 7.616 1.00 86.47C ATOM 125 CD2 LEU A 45 −53.555 19.485 10.008 1.00 82.85 C ATOM 126 NALA A 46 −48.571 19.830 10.330 1.00 78.89 N ATOM 127 CA ALA A 46 −47.78320.933 10.893 1.00 76.32 C ATOM 128 C ALA A 46 −46.639 21.352 9.985 1.0075.76 C ATOM 129 O ALA A 46 −46.453 22.539 9.731 1.00 78.99 O ATOM 130CB ALA A 46 −47.227 20.535 12.244 1.00 75.15 C ATOM 131 N ILE A 47−45.885 20.371 9.505 1.00 72.82 N ATOM 132 CA ILE A 47 −44.751 20.6138.596 1.00 72.71 C ATOM 133 C ILE A 47 −45.206 21.216 7.267 1.00 69.65 CATOM 134 O ILE A 47 −44.651 22.209 6.815 1.00 68.44 O ATOM 135 CB ILE A47 −43.984 19.310 8.290 1.00 73.57 C ATOM 136 CG1 ILE A 47 −43.27618.777 9.540 1.00 73.61 C ATOM 137 CG2 ILE A 47 −42.961 19.534 7.1791.00 73.63 C ATOM 138 CD1 ILE A 47 −42.811 17.358 9.399 1.00 71.65 CATOM 139 N VAL A 48 −46.216 20.606 6.659 1.00 68.32 N ATOM 140 CA VAL A48 −46.721 21.051 5.360 1.00 69.33 C ATOM 141 C VAL A 48 −47.390 22.4405.463 1.00 71.00 C ATOM 142 O VAL A 48 −47.115 23.305 4.638 1.00 72.48 OATOM 143 CB VAL A 48 −47.692 20.011 4.722 1.00 69.55 C ATOM 144 CG1 VALA 48 −48.178 20.489 3.364 1.00 64.85 C ATOM 145 CG2 VAL A 48 −47.00818.642 4.585 1.00 66.28 C ATOM 146 N PHE A 49 −48.248 22.640 6.468 1.0070.84 N ATOM 147 CA PHE A 49 −48.951 23.916 6.654 1.00 69.45 C ATOM 148C PHE A 49 −47.994 25.077 6.803 1.00 71.34 C ATOM 149 O PHE A 49 −48.03926.025 6.017 1.00 71.00 O ATOM 150 CB PHE A 49 −49.869 23.866 7.886 1.0070.88 C ATOM 151 CG PHE A 49 −50.669 25.123 8.103 1.00 71.45 C ATOM 152CD1 PHE A 49 −51.932 25.254 7.554 1.00 72.92 C ATOM 153 CD2 PHE A 49−50.161 26.176 8.855 1.00 75.73 C ATOM 154 CE1 PHE A 49 −52.678 26.4147.751 1.00 73.41 C ATOM 155 CE2 PHE A 49 −50.905 27.336 9.052 1.00 74.53C ATOM 156 CZ PHE A 49 −52.162 27.450 8.498 1.00 69.47 C ATOM 157 N GLYA 50 −47.133 24.985 7.821 1.00 71.43 N ATOM 158 CA GLY A 50 −46.19326.040 8.158 1.00 67.82 C ATOM 159 C GLY A 50 −45.210 26.409 7.066 1.0068.17 C ATOM 160 O GLY A 50 −44.952 27.592 6.852 1.00 67.71 O ATOM 161 NASN A 51 −44.662 25.402 6.381 1.00 66.99 N ATOM 162 CA ASN A 51 −43.71325.636 5.273 1.00 67.98 C ATOM 163 C ASN A 51 −44.414 26.145 3.998 1.0071.16 C ATOM 164 O ASN A 51 −43.847 26.968 3.274 1.00 69.41 O ATOM 165CB ASN A 51 −42.890 24.395 4.997 1.00 68.88 C ATOM 166 CG ASN A 51−41.927 24.083 6.134 1.00 71.66 C ATOM 167 OD1 ASN A 51 −40.914 24.7586.305 1.00 69.08 O ATOM 168 ND2 ASN A 51 −42.241 23.063 6.913 1.00 68.08N ATOM 169 N VAL A 52 −45.631 25.656 3.730 1.00 67.56 N ATOM 170 CA VALA 52 −46.453 26.245 2.687 1.00 70.68 C ATOM 171 C VAL A 52 −46.61627.731 2.994 1.00 71.64 C ATOM 172 O VAL A 52 −46.463 28.558 2.108 1.0072.88 O ATOM 173 CB VAL A 52 −47.860 25.599 2.575 1.00 69.27 C ATOM 174CG1 VAL A 52 −48.837 26.549 1.903 1.00 71.90 C ATOM 175 CG2 VAL A 52−47.784 24.286 1.829 1.00 67.05 C ATOM 176 N LEU A 53 −46.921 28.0494.256 1.00 73.15 N ATOM 177 CA LEU A 53 −47.156 29.441 4.688 1.00 72.57C ATOM 178 C LEU A 53 −45.913 30.335 4.500 1.00 73.72 C ATOM 179 O LEU A53 −46.043 31.494 4.120 1.00 76.55 O ATOM 180 CB LEU A 53 −47.632 29.4826.147 1.00 72.34 C ATOM 181 CG LEU A 53 −48.422 30.705 6.593 1.00 74.13C ATOM 182 CD1 LEU A 53 −49.812 30.716 5.973 1.00 73.24 C ATOM 183 CD2LEU A 53 −48.530 30.738 8.109 1.00 76.12 C ATOM 184 N VAL A 54 −44.72329.789 4.765 1.00 72.68 N ATOM 185 CA VAL A 54 −43.458 30.517 4.563 1.0070.92 C ATOM 186 C VAL A 54 −43.183 30.795 3.076 1.00 72.46 C ATOM 187 OVAL A 54 −42.786 31.903 2.711 1.00 72.67 O ATOM 188 CB VAL A 54 −42.25529.741 5.161 1.00 73.28 C ATOM 189 CG1 VAL A 54 −40.937 30.380 4.7691.00 72.16 C ATOM 190 CG2 VAL A 54 −42.376 29.647 6.682 1.00 69.63 CATOM 191 N ILE A 55 −43.395 29.787 2.233 1.00 69.98 N ATOM 192 CA ILE A55 −43.164 29.906 0.791 1.00 68.58 C ATOM 193 C ILE A 55 −44.076 30.9600.177 1.00 69.05 C ATOM 194 O ILE A 55 −43.606 31.844 −0.552 1.00 65.40O ATOM 195 CB ILE A 55 −43.379 28.569 0.090 1.00 68.25 C ATOM 196 CG1ILE A 55 −42.253 27.591 0.454 1.00 71.45 C ATOM 197 CG2 ILE A 55 −43.43028.766 −1.409 1.00 72.11 C ATOM 198 CD1 ILE A 55 −42.573 26.152 0.1271.00 71.51 C ATOM 199 N THR A 56 −45.369 30.840 0.491 1.00 70.28 N ATOM200 CA THR A 56 −46.403 31.770 0.067 1.00 69.62 C ATOM 201 C THR A 56−46.078 33.211 0.454 1.00 71.19 C ATOM 202 O THR A 56 −46.188 34.089−0.372 1.00 72.89 O ATOM 203 CB THR A 56 −47.780 31.399 0.686 1.00 71.36C ATOM 204 OG1 THR A 56 −48.082 30.019 0.434 1.00 65.43 O ATOM 205 CG2THR A 56 −48.896 32.281 0.110 1.00 69.44 C ATOM 206 N ALA A 57 −45.67833.423 1.714 1.00 71.45 N ATOM 207 CA ALA A 57 −45.368 34.762 2.263 1.0070.84 C ATOM 208 C ALA A 57 −44.180 35.464 1.584 1.00 71.90 C ATOM 209 OALA A 57 −44.220 36.665 1.339 1.00 70.99 O ATOM 210 CB ALA A 57 −45.11934.666 3.767 1.00 67.19 C ATOM 211 N ILE A 58 −43.128 34.712 1.289 1.0071.62 N ATOM 212 CA ILE A 58 −41.960 35.250 0.586 1.00 71.02 C ATOM 213C ILE A 58 −42.294 35.462 −0.910 1.00 76.54 C ATOM 214 O ILE A 58−41.898 36.471 −1.503 1.00 79.19 O ATOM 215 CB ILE A 58 −40.758 34.3210.745 1.00 69.83 C ATOM 216 CG1 ILE A 58 −40.343 34.235 2.227 1.00 69.16C ATOM 217 CG2 ILE A 58 −39.585 34.805 −0.082 1.00 69.14 C ATOM 218 CD1ILE A 58 −39.371 33.129 2.513 1.00 70.35 C ATOM 219 N ALA A 59 −43.01634.511 −1.505 1.00 74.84 N ATOM 220 CA ALA A 59 −43.420 34.599 −2.9131.00 75.72 C ATOM 221 C ALA A 59 −44.422 35.722 −3.174 1.00 76.56 C ATOM222 O ALA A 59 −44.416 36.311 −4.256 1.00 77.62 O ATOM 223 CB ALA A 59−43.999 33.268 −3.385 1.00 72.21 C ATOM 224 N LYS A 60 −45.271 36.012−2.184 1.00 76.98 N ATOM 225 CA LYS A 60 −46.336 37.010 −2.323 1.0076.24 C ATOM 226 C LYS A 60 −45.932 38.438 −1.965 1.00 77.94 C ATOM 227O LYS A 60 −46.282 39.371 −2.694 1.00 75.40 O ATOM 228 CB LYS A 60−47.532 36.605 −1.466 1.00 75.25 C ATOM 229 CG LYS A 60 −48.667 37.578−1.530 1.00 77.75 C ATOM 230 CD LYS A 60 −49.959 36.950 −1.164 1.0078.52 C ATOM 231 CE LYS A 60 −51.028 37.983 −1.189 1.00 79.74 C ATOM 232NZ LYS A 60 −52.299 37.333 −1.081 1.00 83.19 N ATOM 233 N PHE A 61−45.209 38.597 −0.856 1.00 78.18 N ATOM 234 CA PHE A 61 −44.861 39.919−0.334 1.00 79.67 C ATOM 235 C PHE A 61 −43.457 40.379 −0.743 1.00 81.98C ATOM 236 O PHE A 61 −42.443 39.855 −0.253 1.00 78.47 O ATOM 237 CB PHEA 61 −45.055 39.943 1.185 1.00 78.50 C ATOM 238 CG PHE A 61 −46.48839.744 1.589 1.00 77.03 C ATOM 239 CD1 PHE A 61 −46.946 38.513 2.0131.00 74.37 C ATOM 240 CD2 PHE A 61 −47.385 40.799 1.536 1.00 79.08 CATOM 241 CE1 PHE A 61 −48.267 38.334 2.380 1.00 77.21 C ATOM 242 CE2 PHEA 61 −48.708 40.621 1.903 1.00 77.13 C ATOM 243 CZ PHE A 61 −49.14139.382 2.326 1.00 75.36 C ATOM 244 N GLU A 62 −43.450 41.369 −1.648 1.0084.43 N ATOM 245 CA GLU A 62 −42.247 42.031 −2.208 1.00 86.75 C ATOM 246C GLU A 62 −41.256 42.491 −1.122 1.00 89.37 C ATOM 247 O GLU A 62−40.040 42.414 −1.313 1.00 90.84 O ATOM 248 CB GLU A 62 −42.699 43.219−3.080 1.00 86.58 C ATOM 249 CG GLU A 62 −41.641 43.860 −3.967 1.0087.94 C ATOM 250 CD GLU A 62 −42.234 44.940 −4.911 1.00 90.76 C ATOM 251OE1 GLU A 62 −43.321 44.720 −5.505 1.00 96.67 O ATOM 252 OE2 GLU A 62−41.611 46.016 −5.065 1.00 89.94 O ATOM 253 N ARG A 63 −41.798 42.9640.003 1.00 90.60 N ATOM 254 CA ARG A 63 −41.036 43.318 1.207 1.00 88.93C ATOM 255 C ARG A 63 −40.139 42.177 1.705 1.00 88.02 C ATOM 256 O ARG A63 −39.007 42.409 2.131 1.00 88.49 O ATOM 257 CB ARG A 63 −42.028 43.6912.309 1.00 91.49 C ATOM 258 CG ARG A 63 −41.443 43.980 3.698 1.00 95.03C ATOM 259 CD ARG A 63 −42.573 44.340 4.643 1.00 99.84 C ATOM 260 NE ARGA 63 −42.113 44.841 5.939 1.00 103.70 N ATOM 261 CZ ARG A 63 −42.90345.373 6.882 1.00 105.87 C ATOM 262 NH1 ARG A 63 −44.231 45.491 6.7081.00 107.12 N ATOM 263 NH2 ARG A 63 −42.362 45.796 8.028 1.00 109.62 NATOM 264 N LEU A 64 −40.658 40.955 1.648 1.00 85.74 N ATOM 265 CA LEU A64 −39.932 39.775 2.097 1.00 82.11 C ATOM 266 C LEU A 64 −39.032 39.1681.011 1.00 81.55 C ATOM 267 O LEU A 64 −38.378 38.184 1.272 1.00 78.38 OATOM 268 CB LEU A 64 −40.923 38.712 2.602 1.00 80.82 C ATOM 269 CG LEU A64 −41.851 39.090 3.769 1.00 79.80 C ATOM 270 CD1 LEU A 64 −42.89937.999 4.007 1.00 70.96 C ATOM 271 CD2 LEU A 64 −41.056 39.337 5.0381.00 73.56 C ATOM 272 N GLN A 65 −38.992 39.745 −0.198 1.00 82.04 N ATOM273 CA GLN A 65 −38.174 39.189 −1.308 1.00 83.43 C ATOM 274 C GLN A 65−36.720 39.672 −1.264 1.00 83.31 C ATOM 275 O GLN A 65 −36.305 40.518−2.060 1.00 86.84 O ATOM 276 CB GLN A 65 −38.820 39.500 −2.662 1.0082.54 C ATOM 277 CG GLN A 65 −40.120 38.749 −2.856 1.00 84.91 C ATOM 278CD GLN A 65 −40.789 39.016 −4.182 1.00 88.24 C ATOM 279 OE1 GLN A 65−40.516 40.018 −4.857 1.00 97.81 O ATOM 280 NE2 GLN A 65 −41.681 38.114−4.569 1.00 97.75 N ATOM 281 N THR A 66 −35.973 39.113 −0.320 1.00 80.37N ATOM 282 CA THR A 66 −34.561 39.413 −0.090 1.00 79.80 C ATOM 283 C THRA 66 −33.747 38.170 −0.355 1.00 79.26 C ATOM 284 O THR A 66 −34.30637.105 −0.428 1.00 82.12 O ATOM 285 CB THR A 66 −34.334 39.778 1.3531.00 79.66 C ATOM 286 OG1 THR A 66 −34.641 38.639 2.177 1.00 79.38 OATOM 287 CG2 THR A 66 −35.218 40.953 1.753 1.00 75.58 C ATOM 288 N VAL A67 −32.431 38.303 −0.492 1.00 76.86 N ATOM 289 CA VAL A 67 −31.56137.138 −0.771 1.00 76.42 C ATOM 290 C VAL A 67 −31.679 36.080 0.342 1.0076.58 C ATOM 291 O VAL A 67 −31.869 34.888 0.050 1.00 76.26 O ATOM 292CB VAL A 67 −30.087 37.543 −0.956 1.00 77.31 C ATOM 293 CG1 VAL A 67−29.185 36.325 −0.914 1.00 75.37 C ATOM 294 CG2 VAL A 67 −29.900 38.301−2.270 1.00 72.45 C ATOM 295 N THR A 68 −31.568 36.512 1.597 1.00 71.98N ATOM 296 CA THR A 68 −31.754 35.609 2.736 1.00 72.07 C ATOM 297 C THRA 68 −33.028 34.797 2.566 1.00 71.33 C ATOM 298 O THR A 68 −33.00133.564 2.645 1.00 69.01 O ATOM 299 CB THR A 68 −31.810 36.372 4.062 1.0071.58 C ATOM 300 OG1 THR A 68 −30.546 37.009 4.283 1.00 73.28 O ATOM 301CG2 THR A 68 −32.098 35.420 5.213 1.00 71.59 C ATOM 302 N ASN A 69−34.139 35.488 2.321 1.00 71.44 N ATOM 303 CA ASN A 69 −35.438 34.8102.146 1.00 70.87 C ATOM 304 C ASN A 69 −35.534 33.842 0.957 1.00 71.14 CATOM 305 O ASN A 69 −36.425 32.981 0.941 1.00 73.32 O ATOM 306 CB ASN A69 −36.571 35.835 2.123 1.00 68.01 C ATOM 307 CG ASN A 69 −36.794 36.5013.503 1.00 72.26 C ATOM 308 OD1 ASN A 69 −36.311 36.018 4.539 1.00 66.06O ATOM 309 ND2 ASN A 69 −37.525 37.606 3.513 1.00 72.74 N ATOM 310 N TYRA 70 −34.640 33.969 −0.023 1.00 72.21 N ATOM 311 CA TYR A 70 −34.57833.002 −1.133 1.00 73.01 C ATOM 312 C TYR A 70 −33.995 31.695 −0.5991.00 73.34 C ATOM 313 O TYR A 70 −34.479 30.630 −0.942 1.00 77.66 O ATOM314 CB TYR A 70 −33.781 33.529 −2.341 1.00 74.09 C ATOM 315 CG TYR A 70−34.536 34.496 −3.245 1.00 75.84 C ATOM 316 CD1 TYR A 70 −35.265 35.553−2.732 1.00 82.81 C ATOM 317 CD2 TYR A 70 −34.514 34.348 −4.620 1.0084.86 C ATOM 318 CE1 TYR A 70 −35.957 36.444 −3.555 1.00 84.18 C ATOM319 CE2 TYR A 70 −35.200 35.229 −5.453 1.00 85.94 C ATOM 320 CZ TYR A 70−35.921 36.278 −4.918 1.00 84.44 C ATOM 321 OH TYR A 70 −36.603 37.149−5.771 1.00 85.23 O ATOM 322 N PHE A 71 −32.961 31.788 0.238 1.00 75.67N ATOM 323 CA PHE A 71 −32.384 30.609 0.904 1.00 73.91 C ATOM 324 C PHEA 71 −33.360 30.004 1.903 1.00 75.76 C ATOM 325 O PHE A 71 −33.42628.779 2.046 1.00 72.03 O ATOM 326 CB PHE A 71 −31.108 30.949 1.666 1.0074.63 C ATOM 327 CG PHE A 71 −30.024 31.535 0.817 1.00 72.11 C ATOM 328CD1 PHE A 71 −29.584 30.876 −0.311 1.00 69.28 C ATOM 329 CD2 PHE A 71−29.438 32.748 1.158 1.00 70.30 C ATOM 330 CE1 PHE A 71 −28.583 31.413−1.095 1.00 74.96 C ATOM 331 CE2 PHE A 71 −28.449 33.285 0.396 1.0072.37 C ATOM 332 CZ PHE A 71 −28.013 32.613 −0.749 1.00 76.19 C ATOM 333N ILE A 72 −34.111 30.863 2.595 1.00 71.39 N ATOM 334 CA ILE A 72−35.160 30.393 3.485 1.00 71.36 C ATOM 335 C ILE A 72 −36.192 29.5972.692 1.00 73.02 C ATOM 336 O ILE A 72 −36.652 28.546 3.151 1.00 70.62 OATOM 337 CB ILE A 72 −35.868 31.541 4.197 1.00 68.07 C ATOM 338 CG1 ILEA 72 −34.924 32.236 5.203 1.00 79.57 C ATOM 339 CG2 ILE A 72 −37.08931.044 4.929 1.00 67.38 C ATOM 340 CD1 ILE A 72 −34.499 31.384 6.3671.00 74.31 C ATOM 341 N THR A 73 −36.554 30.100 1.506 1.00 70.93 N ATOM342 CA THR A 73 −37.500 29.397 0.644 1.00 71.88 C ATOM 343 C THR A 73−36.937 28.040 0.264 1.00 73.34 C ATOM 344 O THR A 73 −37.663 27.0450.296 1.00 77.13 O ATOM 345 CB THR A 73 −37.840 30.208 −0.618 1.00 73.11C ATOM 346 OG1 THR A 73 −38.491 31.424 −0.228 1.00 71.91 O ATOM 347 CG2THR A 73 −38.747 29.418 −1.548 1.00 72.28 C ATOM 348 N SER A 74 −35.65428.000 −0.091 1.00 73.09 N ATOM 349 CA SER A 74 −34.981 26.740 −0.4091.00 74.48 C ATOM 350 C SER A 74 −35.030 25.775 0.800 1.00 74.98 C ATOM351 O SER A 74 −35.270 24.570 0.638 1.00 78.94 O ATOM 352 CB SER A 74−33.546 27.014 −0.877 1.00 74.59 C ATOM 353 OG SER A 74 −32.820 25.829−1.084 1.00 73.96 O ATOM 354 N LEU A 75 −34.810 26.318 1.995 1.00 73.74N ATOM 355 CA LEU A 75 −34.911 25.567 3.255 1.00 72.08 C ATOM 356 C LEUA 75 −36.351 25.067 3.482 1.00 71.55 C ATOM 357 O LEU A 75 −36.56523.897 3.819 1.00 71.52 O ATOM 358 CB LEU A 75 −34.437 26.455 4.418 1.0072.86 C ATOM 359 CG LEU A 75 −34.085 25.884 5.771 1.00 78.35 C ATOM 360CD1 LEU A 75 −33.026 24.797 5.661 1.00 80.50 C ATOM 361 CD2 LEU A 75−33.614 27.061 6.695 1.00 77.36 C ATOM 362 N ALA A 76 −37.326 25.9583.290 1.00 69.20 N ATOM 363 CA ALA A 76 −38.758 25.610 3.373 1.00 70.43C ATOM 364 C ALA A 76 −39.206 24.520 2.362 1.00 70.45 C ATOM 365 O ALA A76 −40.112 23.747 2.658 1.00 73.21 O ATOM 366 CB ALA A 76 −39.613 26.8643.200 1.00 67.40 C ATOM 367 N CYS A 77 −38.578 24.461 1.184 1.00 72.62 NATOM 368 CA CYS A 77 −38.911 23.411 0.176 1.00 72.54 C ATOM 369 C CYS A77 −38.399 22.039 0.601 1.00 71.97 C ATOM 370 O CYS A 77 −39.084 21.0470.401 1.00 75.84 O ATOM 371 CB CYS A 77 −38.350 23.752 −1.195 1.00 71.94C ATOM 372 SG CYS A 77 −39.135 25.162 −1.970 1.00 78.85 S ATOM 373 N ALAA 78 −37.197 22.000 1.181 1.00 71.07 N ATOM 374 CA ALA A 78 −36.60920.774 1.704 1.00 70.88 C ATOM 375 C ALA A 78 −37.457 20.206 2.846 1.0071.74 C ATOM 376 O ALA A 78 −37.644 18.997 2.947 1.00 72.80 O ATOM 377CB ALA A 78 −35.174 21.031 2.172 1.00 68.59 C ATOM 378 N ASP A 79−37.964 21.083 3.698 1.00 71.88 N ATOM 379 CA ASP A 79 −38.859 20.6764.783 1.00 71.72 C ATOM 380 C ASP A 79 −40.240 20.312 4.244 1.00 70.22 CATOM 381 O ASP A 79 −40.872 19.384 4.743 1.00 70.23 O ATOM 382 CB ASP A79 −38.934 21.775 5.847 1.00 68.87 C ATOM 383 CG ASP A 79 −37.609 21.9316.630 1.00 82.57 C ATOM 384 OD1 ASP A 79 −36.914 20.899 6.889 1.00 83.92O ATOM 385 OD2 ASP A 79 −37.258 23.081 6.992 1.00 79.92 O ATOM 386 N LEUA 80 −40.712 21.034 3.237 1.00 67.28 N ATOM 387 CA LEU A 80 −42.00820.683 2.616 1.00 68.96 C ATOM 388 C LEU A 80 −41.960 19.284 2.009 1.0065.98 C ATOM 389 O LEU A 80 −42.889 18.505 2.184 1.00 70.25 O ATOM 390CB LEU A 80 −42.430 21.705 1.562 1.00 63.48 C ATOM 391 CG LEU A 80−43.828 21.494 0.969 1.00 70.22 C ATOM 392 CD1 LEU A 80 −44.896 21.3462.048 1.00 68.20 C ATOM 393 CD2 LEU A 80 −44.193 22.645 −0.015 1.0074.86 C ATOM 394 N VAL A 81 −40.877 18.972 1.302 1.00 66.65 N ATOM 395CA VAL A 81 −40.686 17.628 0.713 1.00 68.44 C ATOM 396 C VAL A 81−40.559 16.537 1.815 1.00 68.93 C ATOM 397 O VAL A 81 −41.066 15.4261.647 1.00 71.60 O ATOM 398 CB VAL A 81 −39.475 17.608 −0.253 1.00 68.36C ATOM 399 CG1 VAL A 81 −39.202 16.216 −0.740 1.00 65.96 C ATOM 400 CG2VAL A 81 −39.713 18.581 −1.445 1.00 63.74 C ATOM 401 N MET A 82 −39.88916.859 2.923 1.00 67.67 N ATOM 402 CA MET A 82 −39.862 15.968 4.099 1.0069.67 C ATOM 403 C MET A 82 −41.282 15.658 4.578 1.00 67.76 C ATOM 404 OMET A 82 −41.632 14.503 4.804 1.00 66.68 O ATOM 405 CB MET A 82 −39.08816.603 5.245 1.00 70.60 C ATOM 406 CG MET A 82 −37.580 16.550 5.089 1.0076.37 C ATOM 407 SD MET A 82 −36.812 15.024 5.666 1.00 86.09 S ATOM 408CE MET A 82 −37.447 15.024 7.316 1.00 88.14 C ATOM 409 N GLY A 83−42.089 16.703 4.725 1.00 66.27 N ATOM 410 CA GLY A 83 −43.480 16.5645.170 1.00 68.36 C ATOM 411 C GLY A 83 −44.416 15.795 4.250 1.00 71.02 CATOM 412 O GLY A 83 −45.350 15.161 4.730 1.00 72.14 O ATOM 413 N LEU A84 −44.169 15.853 2.939 1.00 71.19 N ATOM 414 CA LEU A 84 −45.010 15.1701.939 1.00 73.59 C ATOM 415 C LEU A 84 −44.469 13.816 1.460 1.00 73.92 CATOM 416 O LEU A 84 −45.224 12.859 1.294 1.00 72.70 O ATOM 417 CB LEU A84 −45.195 16.069 0.702 1.00 72.52 C ATOM 418 CG LEU A 84 −45.901 17.4060.885 1.00 75.09 C ATOM 419 CD1 LEU A 84 −45.933 18.147 −0.445 1.0078.12 C ATOM 420 CD2 LEU A 84 −47.316 17.232 1.430 1.00 77.53 C ATOM 421N ALA A 85 −43.166 13.751 1.237 1.00 73.21 N ATOM 422 CA ALA A 85−42.540 12.584 0.628 1.00 73.60 C ATOM 423 C ALA A 85 −41.858 11.6731.615 1.00 72.66 C ATOM 424 O ALA A 85 −41.825 10.482 1.377 1.00 78.23 OATOM 425 CB ALA A 85 −41.533 13.043 −0.426 1.00 74.36 C ATOM 426 N VAL A86 −41.319 12.218 2.710 1.00 74.33 N ATOM 427 CA VAL A 86 −40.544 11.4193.678 1.00 71.85 C ATOM 428 C VAL A 86 −41.349 11.025 4.910 1.00 71.02 CATOM 429 O VAL A 86 −41.584 9.860 5.132 1.00 73.11 O ATOM 430 CB VAL A86 −39.254 12.136 4.110 1.00 71.51 C ATOM 431 CG1 VAL A 86 −38.40011.213 4.969 1.00 68.65 C ATOM 432 CG2 VAL A 86 −38.467 12.587 2.8851.00 70.81 C ATOM 433 N VAL A 87 −41.773 11.995 5.707 1.00 71.04 N ATOM434 CA VAL A 87 −42.455 11.692 7.002 1.00 71.84 C ATOM 435 C VAL A 87−43.718 10.799 6.915 1.00 72.51 C ATOM 436 O VAL A 87 −43.894 9.9407.783 1.00 76.50 O ATOM 437 CB VAL A 87 −42.798 12.999 7.819 1.00 72.70C ATOM 438 CG1 VAL A 87 −43.506 12.655 9.148 1.00 65.72 C ATOM 439 CG2VAL A 87 −41.549 13.826 8.093 1.00 69.80 C ATOM 440 N PRO A 88 −44.58810.984 5.886 1.00 74.57 N ATOM 441 CA PRO A 88 −45.792 10.131 5.828 1.0073.89 C ATOM 442 C PRO A 88 −45.514 8.629 5.696 1.00 74.31 C ATOM 443 OPRO A 88 −46.204 7.831 6.322 1.00 72.95 O ATOM 444 CB PRO A 88 −46.52910.654 4.580 1.00 72.19 C ATOM 445 CG PRO A 88 −46.040 12.026 4.413 1.0070.91 C ATOM 446 CD PRO A 88 −44.581 11.922 4.748 1.00 75.19 C ATOM 447N PHE A 89 −44.517 8.266 4.895 1.00 75.27 N ATOM 448 CA PHE A 89 −44.1536.857 4.707 1.00 76.82 C ATOM 449 C PHE A 89 −43.325 6.300 5.850 1.0075.58 C ATOM 450 O PHE A 89 −43.466 5.124 6.182 1.00 76.46 O ATOM 451 CBPHE A 89 −43.434 6.665 3.380 1.00 81.35 C ATOM 452 CG PHE A 89 −44.2587.080 2.177 1.00 87.88 C ATOM 453 CD1 PHE A 89 −45.600 6.699 2.046 1.0093.20 C ATOM 454 CD2 PHE A 89 −43.709 7.840 1.186 1.00 87.00 C ATOM 455CE1 PHE A 89 −46.350 7.082 0.940 1.00 93.35 C ATOM 456 CE2 PHE A 89−44.469 8.219 0.076 1.00 92.27 C ATOM 457 CZ PHE A 89 −45.780 7.839−0.040 1.00 91.97 C ATOM 458 N GLY A 90 −42.468 7.125 6.444 1.00 74.71 NATOM 459 CA GLY A 90 −41.738 6.731 7.650 1.00 75.06 C ATOM 460 C GLY A90 −42.728 6.369 8.749 1.00 74.16 C ATOM 461 O GLY A 90 −42.572 5.3419.402 1.00 77.84 O ATOM 462 N ALA A 91 −43.747 7.223 8.939 1.00 72.72 NATOM 463 CA ALA A 91 −44.835 6.994 9.912 1.00 72.43 C ATOM 464 C ALA A91 −45.543 5.664 9.680 1.00 74.22 C ATOM 465 O ALA A 91 −45.668 4.85310.604 1.00 74.73 O ATOM 466 CB ALA A 91 −45.849 8.129 9.849 1.00 69.78C ATOM 467 N ALA A 92 −45.999 5.464 8.437 1.00 73.35 N ATOM 468 CA ALA A92 −46.699 4.241 8.013 1.00 74.78 C ATOM 469 C ALA A 92 −45.850 2.9988.219 1.00 73.86 C ATOM 470 O ALA A 92 −46.355 1.985 8.671 1.00 76.34 OATOM 471 CB ALA A 92 −47.131 4.348 6.541 1.00 73.00 C ATOM 472 N HIS A93 −44.566 3.098 7.881 1.00 75.22 N ATOM 473 CA HIS A 93 −43.592 2.0228.080 1.00 75.74 C ATOM 474 C HIS A 93 −43.521 1.575 9.543 1.00 76.69 CATOM 475 O HIS A 93 −43.429 0.383 9.818 1.00 80.31 O ATOM 476 CB HIS A93 −42.216 2.487 7.579 1.00 77.29 C ATOM 477 CG HIS A 93 −41.151 1.4427.633 1.00 75.35 C ATOM 478 ND1 HIS A 93 −39.816 1.752 7.759 1.00 74.50N ATOM 479 CD2 HIS A 93 −41.220 0.094 7.579 1.00 76.44 C ATOM 480 CE1HIS A 93 −39.110 0.635 7.779 1.00 78.85 C ATOM 481 NE2 HIS A 93 −39.939−0.386 7.671 1.00 74.19 N ATOM 482 N ILE A 94 −43.568 2.533 10.464 1.0077.24 N ATOM 483 CA ILE A 94 −43.534 2.243 11.901 1.00 77.99 C ATOM 484C ILE A 94 −44.855 1.668 12.417 1.00 78.57 C ATOM 485 O ILE A 94 −44.8530.680 13.162 1.00 80.24 O ATOM 486 CB ILE A 94 −43.193 3.506 12.720 1.0078.41 C ATOM 487 CG1 ILE A 94 −41.745 3.911 12.482 1.00 78.28 C ATOM 488CG2 ILE A 94 −43.404 3.262 14.201 1.00 79.28 C ATOM 489 CD1 ILE A 94−41.379 5.210 13.108 1.00 77.39 C ATOM 490 N LEU A 95 −45.967 2.28312.024 1.00 78.45 N ATOM 491 CA LEU A 95 −47.302 1.855 12.478 1.00 79.09C ATOM 492 C LEU A 95 −47.729 0.492 11.925 1.00 80.71 C ATOM 493 O LEU A95 −48.401 −0.271 12.626 1.00 82.74 O ATOM 494 CB LEU A 95 −48.347 2.91612.119 1.00 79.29 C ATOM 495 CG LEU A 95 −48.202 4.255 12.843 1.00 78.03C ATOM 496 CD1 LEU A 95 −49.033 5.334 12.164 1.00 77.81 C ATOM 497 CD2LEU A 95 −48.587 4.110 14.306 1.00 75.72 C ATOM 498 N MET A 96 −47.3460.195 10.685 1.00 80.36 N ATOM 499 CA MET A 96 −47.635 −1.103 10.0661.00 80.57 C ATOM 500 C MET A 96 −46.592 −2.157 10.421 1.00 78.70 C ATOM501 O MET A 96 −46.849 −3.336 10.242 1.00 75.20 O ATOM 502 CB MET A 96−47.727 −0.965 8.552 1.00 80.83 C ATOM 503 CG MET A 96 −48.948 −0.2128.114 1.00 83.20 C ATOM 504 SD MET A 96 −48.947 0.080 6.357 1.00 91.34 SATOM 505 CE MET A 96 −50.698 0.372 6.060 1.00 92.81 C ATOM 506 N LYS A97 −45.429 −1.717 10.918 1.00 80.00 N ATOM 507 CA LYS A 97 −44.302−2.598 11.317 1.00 81.41 C ATOM 508 C LYS A 97 −43.720 −3.385 10.1281.00 79.08 C ATOM 509 O LYS A 97 −43.216 −4.494 10.294 1.00 78.90 O ATOM510 CB LYS A 97 −44.705 −3.535 12.483 1.00 82.51 C ATOM 511 CG LYS A 97−45.084 −2.800 13.750 1.00 85.16 C ATOM 512 CD LYS A 97 −45.946 −3.66314.664 1.00 86.38 C ATOM 513 CE LYS A 97 −46.262 −2.937 15.971 1.0090.74 C ATOM 514 NZ LYS A 97 −46.864 −1.569 15.763 1.00 92.55 N ATOM 515N MET A 98 −43.797 −2.788 8.941 1.00 78.37 N ATOM 516 CA MET A 98−43.317 −3.387 7.703 1.00 78.62 C ATOM 517 C MET A 98 −43.508 −2.3786.592 1.00 78.05 C ATOM 518 O MET A 98 −44.326 −1.458 6.720 1.00 80.11 OATOM 519 CB MET A 98 −44.045 −4.706 7.375 1.00 79.92 C ATOM 520 CG MET A98 −45.573 −4.628 7.326 1.00 84.42 C ATOM 521 SD MET A 98 −46.226 −4.3505.681 1.00 96.30 S ATOM 522 CE MET A 98 −45.936 −5.962 4.938 1.00 95.48C ATOM 523 N TRP A 99 −42.754 −2.552 5.510 1.00 77.49 N ATOM 524 CA TRPA 99 −42.823 −1.681 4.333 1.00 75.69 C ATOM 525 C TRP A 99 −43.856−2.268 3.372 1.00 73.64 C ATOM 526 O TRP A 99 −43.802 −3.441 3.079 1.0077.31 O ATOM 527 CB TRP A 99 −41.444 −1.587 3.691 1.00 74.33 C ATOM 528CG TRP A 99 −41.339 −0.599 2.633 1.00 74.05 C ATOM 529 CD1 TRP A 99−41.293 −0.838 1.306 1.00 70.83 C ATOM 530 CD2 TRP A 99 −41.262 0.8212.788 1.00 73.53 C ATOM 531 NE1 TRP A 99 −41.195 0.331 0.624 1.00 73.91N ATOM 532 CE2 TRP A 99 −41.172 1.368 1.505 1.00 73.77 C ATOM 533 CE3TRP A 99 −41.262 1.680 3.887 1.00 74.17 C ATOM 534 CZ2 TRP A 99 −41.0832.737 1.277 1.00 75.99 C ATOM 535 CZ3 TRP A 99 −41.172 3.054 3.664 1.0071.44 C ATOM 536 CH2 TRP A 99 −41.083 3.564 2.370 1.00 77.11 C ATOM 537N THR A 100 −44.795 −1.451 2.892 1.00 73.03 N ATOM 538 CA THR A 100−45.840 −1.896 1.924 1.00 72.98 C ATOM 539 C THR A 100 −45.766 −1.2360.546 1.00 71.23 C ATOM 540 O THR A 100 −46.562 −1.571 −0.333 1.00 71.17O ATOM 541 CB THR A 100 −47.266 −1.597 2.444 1.00 73.76 C ATOM 542 OG1THR A 100 −47.509 −0.178 2.405 1.00 76.78 O ATOM 543 CG2 THR A 100−47.446 −2.076 3.842 1.00 78.02 C ATOM 544 N PHE A 101 −44.829 −0.3120.355 1.00 70.30 N ATOM 545 CA PHE A 101 −44.748 0.499 −0.870 1.00 72.26C ATOM 546 C PHE A 101 −43.772 −0.083 −1.920 1.00 74.09 C ATOM 547 O PHEA 101 −43.621 0.485 −3.018 1.00 74.48 O ATOM 548 CB PHE A 101 −44.4141.947 −0.481 1.00 72.34 C ATOM 549 CG PHE A 101 −45.268 2.457 0.663 1.0070.71 C ATOM 550 CD1 PHE A 101 −44.766 2.495 1.965 1.00 71.75 C ATOM 551CD2 PHE A 101 −46.572 2.887 0.438 1.00 68.75 C ATOM 552 CE1 PHE A 101−45.552 2.961 3.028 1.00 71.54 C ATOM 553 CE2 PHE A 101 −47.364 3.3541.493 1.00 70.01 C ATOM 554 CZ PHE A 101 −46.853 3.391 2.789 1.00 68.99C ATOM 555 N GLY A 102 −43.122 −1.207 −1.581 1.00 73.14 N ATOM 556 CAGLY A 102 −42.234 −1.922 −2.502 1.00 73.21 C ATOM 557 C GLY A 102−40.821 −1.391 −2.546 1.00 72.86 C ATOM 558 O GLY A 102 −40.552 −0.299−2.091 1.00 72.40 O ATOM 559 N ASN A 103 −39.916 −2.184 −3.114 1.0074.31 N ATOM 560 CA ASN A 103 −38.484 −1.824 −3.165 1.00 74.88 C ATOM561 C ASN A 103 −38.129 −0.574 −3.956 1.00 74.74 C ATOM 562 O ASN A 103−37.251 0.175 −3.515 1.00 76.17 O ATOM 563 CB ASN A 103 −37.626 −2.998−3.690 1.00 74.34 C ATOM 564 CG ASN A 103 −37.420 −4.108 −2.651 1.0074.79 C ATOM 565 OD1 ASN A 103 −37.242 −5.271 −3.012 1.00 76.47 O ATOM566 ND2 ASN A 103 −37.444 −3.753 −1.363 1.00 74.57 N ATOM 567 N PHE A104 −38.782 −0.339 −5.098 1.00 74.65 N ATOM 568 CA PHE A 104 −38.4780.860 −5.884 1.00 75.28 C ATOM 569 C PHE A 104 −38.644 2.100 −5.041 1.0075.71 C ATOM 570 O PHE A 104 −37.710 2.895 −4.906 1.00 77.93 O ATOM 571CB PHE A 104 −39.367 1.020 −7.116 1.00 78.02 C ATOM 572 CG PHE A 104−39.279 2.408 −7.727 1.00 79.37 C ATOM 573 CD1 PHE A 104 −38.129 2.802−8.421 1.00 80.42 C ATOM 574 CD2 PHE A 104 −40.337 3.321 −7.603 1.0081.51 C ATOM 575 CE1 PHE A 104 −38.031 4.072 −8.984 1.00 79.83 C ATOM576 CE2 PHE A 104 −40.248 4.598 −8.164 1.00 83.42 C ATOM 577 CZ PHE A104 −39.091 4.975 −8.857 1.00 82.52 C ATOM 578 N TRP A 105 −39.839 2.255−4.478 1.00 76.21 N ATOM 579 CA TRP A 105 −40.134 3.416 −3.670 1.0075.91 C ATOM 580 C TRP A 105 −39.295 3.456 −2.357 1.00 76.37 C ATOM 581O TRP A 105 −38.972 4.534 −1.899 1.00 74.11 O ATOM 582 CB TRP A 105−41.645 3.562 −3.386 1.00 78.05 C ATOM 583 CG TRP A 105 −41.870 4.799−2.623 1.00 82.40 C ATOM 584 CD1 TRP A 105 −42.157 4.889 −1.318 1.0083.43 C ATOM 585 CD2 TRP A 105 −41.810 6.147 −3.120 1.00 87.60 C ATOM586 NE1 TRP A 105 −42.288 6.182 −0.950 1.00 83.52 N ATOM 587 CE2 TRP A105 −42.082 6.994 −2.033 1.00 84.67 C ATOM 588 CE3 TRP A 105 −41.5566.716 −4.381 1.00 86.42 C ATOM 589 CZ2 TRP A 105 −42.109 8.401 −2.1551.00 81.05 C ATOM 590 CZ3 TRP A 105 −41.583 8.115 −4.507 1.00 84.49 CATOM 591 CH2 TRP A 105 −41.860 8.938 −3.391 1.00 85.45 C ATOM 592 N CYSA 106 −38.952 2.302 −1.775 1.00 74.15 N ATOM 593 CA CYS A 106 −38.0862.278 −0.573 1.00 75.23 C ATOM 594 C CYS A 106 −36.763 2.982 −0.834 1.0074.63 C ATOM 595 O CYS A 106 −36.332 3.831 −0.045 1.00 72.22 O ATOM 596CB CYS A 106 −37.827 0.848 −0.097 1.00 75.82 C ATOM 597 SG CYS A 106−36.530 0.694 1.167 1.00 75.27 S ATOM 598 N GLU A 107 −36.137 2.618−1.946 1.00 74.18 N ATOM 599 CA GLU A 107 −34.882 3.219 −2.380 1.0073.95 C ATOM 600 C GLU A 107 −35.054 4.698 −2.773 1.00 73.77 C ATOM 601O GLU A 107 −34.185 5.521 −2.469 1.00 69.11 O ATOM 602 CB GLU A 107−34.337 2.411 −3.542 1.00 74.75 C ATOM 603 CG GLU A 107 −33.001 2.841−4.117 1.00 78.43 C ATOM 604 CD GLU A 107 −32.677 2.072 −5.397 1.0082.58 C ATOM 605 OE1 GLU A 107 −32.915 0.843 −5.452 1.00 87.52 O ATOM606 OE2 GLU A 107 −32.186 2.688 −6.352 1.00 90.13 O ATOM 607 N PHE A 108−36.166 5.025 −3.445 1.00 72.86 N ATOM 608 CA PHE A 108 −36.458 6.414−3.816 1.00 70.73 C ATOM 609 C PHE A 108 −36.705 7.236 −2.556 1.00 68.58C ATOM 610 O PHE A 108 −36.073 8.275 −2.357 1.00 66.92 O ATOM 611 CB PHEA 108 −37.676 6.506 −4.729 1.00 70.86 C ATOM 612 CG PHE A 108 −37.7447.783 −5.526 1.00 72.80 C ATOM 613 CD1 PHE A 108 −37.339 7.799 −6.8681.00 78.82 C ATOM 614 CD2 PHE A 108 −38.205 8.965 −4.961 1.00 74.96 CATOM 615 CE1 PHE A 108 −37.396 8.977 −7.621 1.00 76.80 C ATOM 616 CE2PHE A 108 −38.262 10.149 −5.720 1.00 74.68 C ATOM 617 CZ PHE A 108−37.858 10.148 −7.043 1.00 76.41 C ATOM 618 N TRP A 109 −37.628 6.752−1.723 1.00 66.71 N ATOM 619 CA TRP A 109 −37.960 7.358 −0.431 1.0068.89 C ATOM 620 C TRP A 109 −36.701 7.635 0.361 1.00 69.04 C ATOM 621 OTRP A 109 −36.504 8.752 0.807 1.00 68.71 O ATOM 622 CB TRP A 109 −38.8666.437 0.371 1.00 71.86 C ATOM 623 CG TRP A 109 −39.102 6.855 1.747 1.0070.49 C ATOM 624 CD1 TRP A 109 −39.862 7.878 2.149 1.00 72.33 C ATOM 625CD2 TRP A 109 −38.569 6.248 2.938 1.00 74.86 C ATOM 626 NE1 TRP A 109−39.847 7.967 3.516 1.00 76.09 N ATOM 627 CE2 TRP A 109 −39.062 6.9794.025 1.00 72.44 C ATOM 628 CE3 TRP A 109 −37.720 5.156 3.187 1.00 77.42C ATOM 629 CZ2 TRP A 109 −38.747 6.669 5.347 1.00 75.85 C ATOM 630 CZ3TRP A 109 −37.404 4.849 4.532 1.00 74.21 C ATOM 631 CH2 TRP A 109−37.919 5.602 5.575 1.00 73.66 C ATOM 632 N THR A 110 −35.861 6.6070.518 1.00 64.06 N ATOM 633 CA THR A 110 −34.570 6.747 1.190 1.00 66.65C ATOM 634 C THR A 110 −33.707 7.863 0.621 1.00 68.41 C ATOM 635 O THR A110 −33.116 8.624 1.385 1.00 71.70 O ATOM 636 CB THR A 110 −33.757 5.4771.102 1.00 66.89 C ATOM 637 OG1 THR A 110 −34.491 4.404 1.699 1.00 70.60O ATOM 638 CG2 THR A 110 −32.427 5.645 1.830 1.00 64.09 C ATOM 639 N SERA 111 −33.636 7.959 −0.709 1.00 69.98 N ATOM 640 CA SER A 111 −32.8499.017 −1.387 1.00 70.65 C ATOM 641 C SER A 111 −33.343 10.443 −1.0921.00 73.21 C ATOM 642 O SER A 111 −32.524 11.356 −0.899 1.00 76.91 OATOM 643 CB SER A 111 −32.825 8.802 −2.909 1.00 72.35 C ATOM 644 OG SERA 111 −32.247 7.565 −3.262 1.00 69.46 O ATOM 645 N ILE A 112 −34.66910.626 −1.063 1.00 72.31 N ATOM 646 CA ILE A 112 −35.290 11.938 −0.7861.00 70.06 C ATOM 647 C ILE A 112 −35.011 12.365 0.650 1.00 69.36 C ATOM648 O ILE A 112 −34.706 13.535 0.916 1.00 68.07 O ATOM 649 CB ILE A 112−36.819 11.916 −1.006 1.00 68.74 C ATOM 650 CG1 ILE A 112 −37.174 11.632−2.476 1.00 75.90 C ATOM 651 CG2 ILE A 112 −37.426 13.234 −0.645 1.0072.66 C ATOM 652 CD1 ILE A 112 −38.665 11.518 −2.704 1.00 73.21 C ATOM653 N ASP A 113 −35.126 11.403 1.557 1.00 68.09 N ATOM 654 CA ASP A 113−34.823 11.584 2.973 1.00 69.93 C ATOM 655 C ASP A 113 −33.435 12.2183.102 1.00 68.24 C ATOM 656 O ASP A 113 −33.311 13.328 3.600 1.00 69.08O ATOM 657 CB ASP A 113 −34.916 10.225 3.681 1.00 71.22 C ATOM 658 CGASP A 113 −35.001 10.325 5.163 1.00 71.94 C ATOM 659 OD1 ASP A 113−34.453 11.263 5.771 1.00 73.99 O ATOM 660 OD2 ASP A 113 −35.644 9.4195.727 1.00 78.39 O ATOM 661 N VAL A 114 −32.415 11.501 2.640 1.00 69.76N ATOM 662 CA VAL A 114 −31.016 11.951 2.687 1.00 69.79 C ATOM 663 C VALA 114 −30.790 13.289 1.934 1.00 70.38 C ATOM 664 O VAL A 114 −30.02214.132 2.409 1.00 66.61 O ATOM 665 CB VAL A 114 −30.070 10.827 2.1581.00 71.48 C ATOM 666 CG1 VAL A 114 −28.657 11.338 1.891 1.00 71.98 CATOM 667 CG2 VAL A 114 −30.054 9.643 3.147 1.00 68.67 C ATOM 668 N LEU A115 −31.455 13.467 0.780 1.00 69.66 N ATOM 669 CA LEU A 115 −31.35614.717 −0.015 1.00 70.60 C ATOM 670 C LEU A 115 −31.817 15.929 0.7731.00 72.08 C ATOM 671 O LEU A 115 −31.084 16.909 0.895 1.00 71.75 O ATOM672 CB LEU A 115 −32.193 14.621 −1.314 1.00 73.84 C ATOM 673 CG LEU A115 −32.357 15.859 −2.238 1.00 73.55 C ATOM 674 CD1 LEU A 115 −31.01416.399 −2.735 1.00 72.28 C ATOM 675 CD2 LEU A 115 −33.278 15.542 −3.4311.00 66.35 C ATOM 676 N CYS A 116 −33.039 15.841 1.297 1.00 72.44 N ATOM677 CA CYS A 116 −33.670 16.941 2.034 1.00 73.77 C ATOM 678 C CYS A 116−32.868 17.364 3.251 1.00 74.35 C ATOM 679 O CYS A 116 −32.711 18.5703.492 1.00 75.14 O ATOM 680 CB CYS A 116 −35.112 16.586 2.437 1.00 72.52C ATOM 681 SG CYS A 116 −36.268 16.557 1.013 1.00 80.97 S ATOM 682 N VALA 117 −32.365 16.391 4.010 1.00 72.36 N ATOM 683 CA VAL A 117 −31.50916.695 5.167 1.00 73.25 C ATOM 684 C VAL A 117 −30.188 17.351 4.725 1.0075.09 C ATOM 685 O VAL A 117 −29.729 18.311 5.369 1.00 76.15 O ATOM 686CB VAL A 117 −31.217 15.439 6.020 1.00 73.55 C ATOM 687 CG1 VAL A 117−30.199 15.737 7.098 1.00 69.89 C ATOM 688 CG2 VAL A 117 −32.503 14.9166.641 1.00 73.99 C ATOM 689 N THR A 118 −29.592 16.837 3.635 1.00 71.84N ATOM 690 CA THR A 118 −28.329 17.366 3.096 1.00 71.32 C ATOM 691 C THRA 118 −28.501 18.788 2.575 1.00 73.72 C ATOM 692 O THR A 118 −27.71019.656 2.916 1.00 78.08 O ATOM 693 CB THR A 118 −27.767 16.482 1.9491.00 72.20 C ATOM 694 OG1 THR A 118 −27.512 15.161 2.433 1.00 75.03 OATOM 695 CG2 THR A 118 −26.471 17.066 1.389 1.00 65.55 C ATOM 696 N ALAA 119 −29.534 19.013 1.752 1.00 72.84 N ATOM 697 CA ALA A 119 −29.85720.351 1.220 1.00 71.95 C ATOM 698 C ALA A 119 −30.142 21.361 2.334 1.0071.48 C ATOM 699 O ALA A 119 −29.750 22.521 2.226 1.00 73.66 O ATOM 700CB ALA A 119 −31.054 20.284 0.243 1.00 69.50 C ATOM 701 N SER A 120−30.818 20.920 3.394 1.00 71.79 N ATOM 702 CA SER A 120 −31.124 21.8034.531 1.00 71.94 C ATOM 703 C SER A 120 −29.861 22.374 5.153 1.00 73.64C ATOM 704 O SER A 120 −29.724 23.598 5.252 1.00 74.21 O ATOM 705 CB SERA 120 −31.927 21.071 5.584 1.00 71.95 C ATOM 706 OG SER A 120 −33.20220.758 5.077 1.00 75.41 O ATOM 707 N ILE A 121 −28.945 21.500 5.562 1.0074.66 N ATOM 708 CA ILE A 121 −27.694 21.946 6.223 1.00 72.09 C ATOM 709C ILE A 121 −26.767 22.684 5.253 1.00 74.47 C ATOM 710 O ILE A 121−26.066 23.606 5.668 1.00 72.06 O ATOM 711 CB ILE A 121 −26.922 20.7826.903 1.00 72.33 C ATOM 712 CG1 ILE A 121 −25.776 21.297 7.823 1.0075.07 C ATOM 713 CG2 ILE A 121 −26.349 19.826 5.858 1.00 69.34 C ATOM714 CD1 ILE A 121 −26.149 22.421 8.816 1.00 75.41 C ATOM 715 N GLU A 122−26.758 22.289 3.977 1.00 71.61 N ATOM 716 CA GLU A 122 −25.957 23.0132.976 1.00 73.61 C ATOM 717 C GLU A 122 −26.477 24.430 2.815 1.00 71.97C ATOM 718 O GLU A 122 −25.684 25.357 2.719 1.00 73.76 O ATOM 719 CB GLUA 122 −25.936 22.298 1.619 1.00 71.23 C ATOM 720 CG GLU A 122 −25.04221.050 1.616 1.00 78.82 C ATOM 721 CD GLU A 122 −24.898 20.393 0.2491.00 76.68 C ATOM 722 OE1 GLU A 122 −25.210 21.034 −0.777 1.00 81.34 OATOM 723 OE2 GLU A 122 −24.464 19.221 0.208 1.00 82.37 O ATOM 724 N THRA 123 −27.804 24.585 2.788 1.00 71.94 N ATOM 725 CA THR A 123 −28.43725.916 2.693 1.00 71.90 C ATOM 726 C THR A 123 −28.141 26.762 3.909 1.0071.32 C ATOM 727 O THR A 123 −27.722 27.903 3.768 1.00 70.52 O ATOM 728CB THR A 123 −29.940 25.819 2.596 1.00 69.91 C ATOM 729 OG1 THR A 123−30.275 24.924 1.534 1.00 70.66 O ATOM 730 CG2 THR A 123 −30.539 27.2142.354 1.00 68.19 C ATOM 731 N LEU A 124 −28.364 26.197 5.099 1.00 69.53N ATOM 732 CA LEU A 124 −28.044 26.877 6.330 1.00 70.49 C ATOM 733 C LEUA 124 −26.624 27.353 6.294 1.00 69.98 C ATOM 734 O LEU A 124 −26.34928.465 6.684 1.00 72.08 O ATOM 735 CB LEU A 124 −28.230 25.963 7.5611.00 72.66 C ATOM 736 CG LEU A 124 −29.654 25.655 7.991 1.00 71.44 CATOM 737 CD1 LEU A 124 −29.643 24.635 9.128 1.00 74.54 C ATOM 738 CD2LEU A 124 −30.358 26.930 8.384 1.00 68.04 C ATOM 739 N CYS A 125 −25.72626.509 5.822 1.00 72.50 N ATOM 740 CA CYS A 125 −24.319 26.880 5.7271.00 75.56 C ATOM 741 C CYS A 125 −24.087 28.030 4.746 1.00 73.80 C ATOM742 O CYS A 125 −23.222 28.868 4.985 1.00 74.28 O ATOM 743 CB CYS A 125−23.474 25.682 5.322 1.00 76.67 C ATOM 744 SG CYS A 125 −21.781 25.9575.620 1.00 84.21 S ATOM 745 N VAL A 126 −24.860 28.059 3.652 1.00 76.58N ATOM 746 CA VAL A 126 −24.803 29.161 2.654 1.00 74.32 C ATOM 747 C VALA 126 −25.327 30.458 3.269 1.00 74.06 C ATOM 748 O VAL A 126 −24.82031.529 2.959 1.00 74.99 O ATOM 749 CB VAL A 126 −25.612 28.848 1.3811.00 73.89 C ATOM 750 CG1 VAL A 126 −25.614 30.050 0.435 1.00 78.42 CATOM 751 CG2 VAL A 126 −25.056 27.612 0.682 1.00 75.52 C ATOM 752 N ILEA 127 −26.341 30.352 4.138 1.00 73.19 N ATOM 753 CA ILE A 127 −26.90231.527 4.840 1.00 71.30 C ATOM 754 C ILE A 127 −25.846 32.137 5.724 1.0069.71 C ATOM 755 O ILE A 127 −25.706 33.342 5.750 1.00 73.08 O ATOM 756CB ILE A 127 −28.150 31.200 5.671 1.00 70.82 C ATOM 757 CG1 ILE A 127−29.301 30.720 4.782 1.00 74.13 C ATOM 758 CG2 ILE A 127 −28.636 32.4296.410 1.00 72.22 C ATOM 759 CD1 ILE A 127 −30.450 30.161 5.540 1.0073.97 C ATOM 760 N ALA A 128 −25.105 31.298 6.440 1.00 70.03 N ATOM 761CA ALA A 128 −24.031 31.756 7.323 1.00 70.78 C ATOM 762 C ALA A 128−22.929 32.481 6.577 1.00 70.23 C ATOM 763 O ALA A 128 −22.493 33.5447.004 1.00 70.16 O ATOM 764 CB ALA A 128 −23.443 30.585 8.092 1.00 72.00C ATOM 765 N VAL A 129 −22.480 31.905 5.463 1.00 73.67 N ATOM 766 CA VALA 129 −21.415 32.529 4.657 1.00 70.68 C ATOM 767 C VAL A 129 −21.92333.853 4.062 1.00 70.47 C ATOM 768 O VAL A 129 −21.212 34.853 4.092 1.0073.60 O ATOM 769 CB VAL A 129 −20.878 31.595 3.556 1.00 70.74 C ATOM 770CG1 VAL A 129 −19.835 32.328 2.689 1.00 65.41 C ATOM 771 CG2 VAL A 129−20.285 30.331 4.172 1.00 66.69 C ATOM 772 N ASP A 130 −23.149 33.8363.536 1.00 72.22 N ATOM 773 CA ASP A 130 −23.812 35.034 2.993 1.00 69.02C ATOM 774 C ASP A 130 −23.965 36.125 4.036 1.00 68.79 C ATOM 775 O ASPA 130 −23.668 37.270 3.758 1.00 66.69 O ATOM 776 CB ASP A 130 −25.19534.686 2.460 1.00 71.35 C ATOM 777 CG ASP A 130 −25.925 35.902 1.9161.00 78.10 C ATOM 778 OD1 ASP A 130 −25.369 36.570 1.008 1.00 76.18 OATOM 779 OD2 ASP A 130 −27.049 36.181 2.399 1.00 90.58 O ATOM 780 N ARGA 131 −24.434 35.770 5.243 1.00 69.55 N ATOM 781 CA ARG A 131 −24.52036.762 6.342 1.00 67.36 C ATOM 782 C ARG A 131 −23.143 37.268 6.778 1.0067.63 C ATOM 783 O ARG A 131 −23.021 38.445 7.169 1.00 65.43 O ATOM 784CB ARG A 131 −25.291 36.222 7.544 1.00 67.67 C ATOM 785 CG ARG A 131−26.791 35.942 7.317 1.00 70.59 C ATOM 786 CD ARG A 131 −27.581 37.2087.051 1.00 78.70 C ATOM 787 NE ARG A 131 −27.386 37.707 5.685 1.00 80.63N ATOM 788 CZ ARG A 131 −27.690 38.934 5.244 1.00 84.09 C ATOM 789 NH1ARG A 131 −28.227 39.861 6.051 1.00 88.30 N ATOM 790 NH2 ARG A 131−27.454 39.237 3.969 1.00 81.21 N ATOM 791 N TYR A 132 −22.112 36.4146.716 1.00 66.25 N ATOM 792 CA TYR A 132 −20.772 36.872 7.091 1.00 66.91C ATOM 793 C TYR A 132 −20.300 37.951 6.123 1.00 69.24 C ATOM 794 O TYRA 132 −19.816 38.995 6.553 1.00 74.15 O ATOM 795 CB TYR A 132 −19.74435.746 7.153 1.00 64.41 C ATOM 796 CG TYR A 132 −18.362 36.288 7.4661.00 65.53 C ATOM 797 CD1 TYR A 132 −17.989 36.601 8.780 1.00 73.29 CATOM 798 CD2 TYR A 132 −17.433 36.492 6.460 1.00 66.44 C ATOM 799 CE1TYR A 132 −16.716 37.102 9.068 1.00 69.45 C ATOM 800 CE2 TYR A 132−16.170 36.989 6.731 1.00 66.23 C ATOM 801 CZ TYR A 132 −15.809 37.2938.025 1.00 71.49 C ATOM 802 OH TYR A 132 −14.542 37.787 8.266 1.00 69.82O ATOM 803 N PHE A 133 −20.435 37.715 4.827 1.00 69.93 N ATOM 804 CA PHEA 133 −20.010 38.759 3.860 1.00 70.80 C ATOM 805 C PHE A 133 −20.92039.992 3.920 1.00 69.98 C ATOM 806 O PHE A 133 −20.427 41.103 3.794 1.0070.63 O ATOM 807 CB PHE A 133 −19.846 38.184 2.456 1.00 67.77 C ATOM 808CG PHE A 133 −18.650 37.278 2.342 1.00 69.98 C ATOM 809 CD1 PHE A 133−17.352 37.802 2.445 1.00 72.82 C ATOM 810 CD2 PHE A 133 −18.799 35.9142.140 1.00 69.69 C ATOM 811 CE1 PHE A 133 −16.228 36.981 2.345 1.0070.61 C ATOM 812 CE2 PHE A 133 −17.667 35.081 2.039 1.00 71.02 C ATOM813 CZ PHE A 133 −16.385 35.620 2.143 1.00 68.76 C ATOM 814 N ALA A 134−22.221 39.790 4.120 1.00 69.44 N ATOM 815 CA ALA A 134 −23.141 40.9114.273 1.00 68.56 C ATOM 816 C ALA A 134 −22.724 41.816 5.416 1.00 68.68C ATOM 817 O ALA A 134 −22.633 43.022 5.229 1.00 71.94 O ATOM 818 CB ALAA 134 −24.562 40.422 4.491 1.00 67.16 C ATOM 819 N ILE A 135 −22.46541.241 6.589 1.00 68.84 N ATOM 820 CA ILE A 135 −22.145 42.039 7.7921.00 70.46 C ATOM 821 C ILE A 135 −20.732 42.639 7.824 1.00 73.76 C ATOM822 O ILE A 135 −20.483 43.567 8.604 1.00 77.85 O ATOM 823 CB ILE A 135−22.361 41.205 9.107 1.00 68.09 C ATOM 824 CG1 ILE A 135 −22.694 42.12210.290 1.00 70.69 C ATOM 825 CG2 ILE A 135 −21.151 40.315 9.387 1.0054.90 C ATOM 826 CD1 ILE A 135 −23.130 41.371 11.531 1.00 69.39 C ATOM827 N THR A 136 −19.819 42.123 6.998 1.00 72.13 N ATOM 828 CA THR A 136−18.450 42.658 6.911 1.00 72.48 C ATOM 829 C THR A 136 −18.272 43.6025.722 1.00 73.00 C ATOM 830 O THR A 136 −17.205 44.194 5.560 1.00 76.38O ATOM 831 CB THR A 136 −17.408 41.523 6.791 1.00 71.33 C ATOM 832 OG1THR A 136 −17.656 40.755 5.612 1.00 69.78 O ATOM 833 CG2 THR A 136−17.478 40.622 7.980 1.00 76.96 C ATOM 834 N SER A 137 −19.312 43.7434.890 1.00 73.57 N ATOM 835 CA SER A 137 −19.224 44.546 3.673 1.00 71.50C ATOM 836 C SER A 137 −19.370 46.004 4.020 1.00 70.30 C ATOM 837 O SERA 137 −20.079 46.302 4.923 1.00 72.29 O ATOM 838 CB SER A 137 −20.32744.159 2.678 1.00 70.57 C ATOM 839 OG SER A 137 −20.402 45.088 1.6121.00 73.10 O ATOM 840 N PRO A 138 −18.693 46.924 3.292 1.00 73.40 N ATOM841 CA PRO A 138 −18.908 48.362 3.552 1.00 71.10 C ATOM 842 C PRO A 138−20.342 48.823 3.196 1.00 72.24 C ATOM 843 O PRO A 138 −20.791 49.8453.689 1.00 73.27 O ATOM 844 CB PRO A 138 −17.875 49.042 2.656 1.00 72.76C ATOM 845 CG PRO A 138 −16.952 47.966 2.220 1.00 75.11 C ATOM 846 CDPRO A 138 −17.713 46.719 2.207 1.00 70.99 C ATOM 847 N PHE A 139 −21.03048.053 2.343 1.00 75.02 N ATOM 848 CA PHE A 139 −22.436 48.228 2.0121.00 75.94 C ATOM 849 C PHE A 139 −23.097 47.194 2.924 1.00 81.11 C ATOM850 O PHE A 139 −23.703 46.209 2.451 1.00 82.84 O ATOM 851 CB PHE A 139−22.686 47.948 0.517 1.00 70.64 C ATOM 852 CG PHE A 139 −21.537 48.366−0.376 1.00 68.22 C ATOM 853 CD1 PHE A 139 −20.705 47.417 −0.943 1.0064.99 C ATOM 854 CD2 PHE A 139 −21.291 49.686 −0.634 1.00 54.92 C ATOM855 CE1 PHE A 139 −19.676 47.789 −1.736 1.00 53.34 C ATOM 856 CE2 PHE A139 −20.261 50.061 −1.427 1.00 55.02 C ATOM 857 CZ PHE A 139 −19.44849.112 −1.982 1.00 59.66 C ATOM 858 N LYS A 140 −22.970 47.430 4.2391.00 82.78 N ATOM 859 CA LYS A 140 −23.326 46.437 5.288 1.00 86.29 CATOM 860 C LYS A 140 −24.732 45.890 5.161 1.00 84.99 C ATOM 861 O LYS A140 −25.668 46.622 4.835 1.00 84.00 O ATOM 862 CB LYS A 140 −23.14447.003 6.710 1.00 86.05 C ATOM 863 CG LYS A 140 −21.716 47.263 7.1041.00 93.56 C ATOM 864 CD LYS A 140 −21.533 47.754 8.540 1.00 92.32 CATOM 865 CE LYS A 140 −20.068 48.178 8.748 1.00 94.63 C ATOM 866 NZ LYSA 140 −19.803 48.731 10.115 1.00 103.84 N ATOM 867 N TYR A 141 −24.83644.589 5.424 1.00 85.69 N ATOM 868 CA TYR A 141 −26.070 43.783 5.3831.00 87.34 C ATOM 869 C TYR A 141 −26.642 43.451 3.984 1.00 87.45 C ATOM870 O TYR A 141 −27.393 42.477 3.859 1.00 87.44 O ATOM 871 CB TYR A 141−27.154 44.369 6.294 1.00 89.48 C ATOM 872 CG TYR A 141 −26.773 44.2547.729 1.00 86.02 C ATOM 873 CD1 TYR A 141 −27.055 43.095 8.434 1.0082.79 C ATOM 874 CD2 TYR A 141 −26.132 45.293 8.380 1.00 87.79 C ATOM875 CE1 TYR A 141 −26.717 42.968 9.747 1.00 95.42 C ATOM 876 CE2 TYR A141 −25.777 45.189 9.716 1.00 94.16 C ATOM 877 CZ TYR A 141 −26.07144.020 10.400 1.00 97.24 C ATOM 878 OH TYR A 141 −25.728 43.897 11.7201.00 91.49 O ATOM 879 N GLN A 142 −26.313 44.220 2.958 1.00 83.88 N ATOM880 CA GLN A 142 −26.686 43.826 1.640 1.00 87.53 C ATOM 881 C GLN A 142−25.823 42.632 1.368 1.00 85.35 C ATOM 882 O GLN A 142 −24.602 42.6821.609 1.00 85.83 O ATOM 883 CB GLN A 142 −26.375 44.883 0.590 1.00 86.73C ATOM 884 CG GLN A 142 −27.186 46.134 0.674 1.00 87.65 C ATOM 885 CDGLN A 142 −26.769 47.145 −0.388 1.00 91.57 C ATOM 886 OE1 GLN A 142−26.382 46.780 −1.508 1.00 90.57 O ATOM 887 NE2 GLN A 142 −26.847 48.425−0.040 1.00 99.82 N ATOM 888 N SER A 143 −26.446 41.562 0.879 1.00 81.37N ATOM 889 CA SER A 143 −25.716 40.420 0.415 1.00 78.53 C ATOM 890 C SERA 143 −24.884 40.866 −0.772 1.00 77.76 C ATOM 891 O SER A 143 −25.32041.731 −1.554 1.00 76.92 O ATOM 892 CB SER A 143 −26.662 39.315 −0.0301.00 79.11 C ATOM 893 OG SER A 143 −25.960 38.321 −0.743 1.00 79.05 OATOM 894 N LEU A 144 −23.695 40.284 −0.913 1.00 75.59 N ATOM 895 CA LEUA 144 −22.854 40.541 −2.082 1.00 78.96 C ATOM 896 C LEU A 144 −23.46139.937 −3.371 1.00 77.36 C ATOM 897 O LEU A 144 −23.012 40.278 −4.4771.00 79.84 O ATOM 898 CB LEU A 144 −21.433 40.007 −1.867 1.00 80.08 CATOM 899 CG LEU A 144 −20.660 40.517 −0.639 1.00 82.77 C ATOM 900 CD1LEU A 144 −19.218 40.045 −0.740 1.00 84.51 C ATOM 901 CD2 LEU A 144−20.714 42.055 −0.474 1.00 77.04 C ATOM 902 N LEU A 145 −24.466 39.056−3.210 1.00 74.06 N ATOM 903 CA LEU A 145 −25.162 38.394 −4.293 1.0075.14 C ATOM 904 C LEU A 145 −26.417 39.116 −4.759 1.00 74.57 C ATOM 905O LEU A 145 −27.158 39.675 −3.951 1.00 76.73 O ATOM 906 CB LEU A 145−25.610 36.999 −3.836 1.00 74.59 C ATOM 907 CG LEU A 145 −24.572 36.016−3.326 1.00 73.05 C ATOM 908 CD1 LEU A 145 −25.249 34.715 −2.973 1.0075.64 C ATOM 909 CD2 LEU A 145 −23.502 35.785 −4.364 1.00 79.94 C ATOM910 N THR A 146 −26.645 39.095 −6.070 1.00 74.52 N ATOM 911 CA THR A 146−27.901 39.539 −6.654 1.00 76.04 C ATOM 912 C THR A 146 −28.928 38.413−6.482 1.00 75.70 C ATOM 913 O THR A 146 −28.574 37.282 −6.104 1.0075.21 O ATOM 914 CB THR A 146 −27.772 39.850 −8.156 1.00 76.46 C ATOM915 OG1 THR A 146 −27.440 38.650 −8.860 1.00 79.82 O ATOM 916 CG2 THR A146 −26.702 40.917 −8.423 1.00 71.60 C ATOM 917 N LYS A 147 −30.19338.719 −6.760 1.00 77.16 N ATOM 918 CA LYS A 147 −31.279 37.718 −6.6871.00 77.11 C ATOM 919 C LYS A 147 −30.999 36.539 −7.633 1.00 76.19 CATOM 920 O LYS A 147 −31.219 35.383 −7.267 1.00 73.96 O ATOM 921 CB LYSA 147 −32.646 38.315 −7.052 1.00 78.39 C ATOM 922 CG LYS A 147 −33.10739.543 −6.264 1.00 81.83 C ATOM 923 CD LYS A 147 −33.377 39.263 −4.8151.00 87.01 C ATOM 924 CE LYS A 147 −33.758 40.559 −4.072 1.00 89.91 CATOM 925 NZ LYS A 147 −35.048 41.155 −4.556 1.00 91.41 N ATOM 926 N ASNA 148 −30.512 36.843 −8.843 1.00 76.47 N ATOM 927 CA ASN A 148 −30.19935.809 −9.849 1.00 77.14 C ATOM 928 C ASN A 148 −29.023 34.912 −9.4761.00 76.20 C ATOM 929 O ASN A 148 −29.113 33.689 −9.647 1.00 74.96 OATOM 930 CB ASN A 148 −29.958 36.431 −11.244 1.00 79.53 C ATOM 931 CGASN A 148 −31.254 36.824 −11.956 1.00 84.32 C ATOM 932 OD1 ASN A 148−32.355 36.395 −11.586 1.00 89.19 O ATOM 933 ND2 ASN A 148 −31.12137.648 −12.993 1.00 85.88 N ATOM 934 N LYS A 149 −27.933 35.503 −8.9761.00 73.79 N ATOM 935 CA LYS A 149 −26.801 34.706 −8.474 1.00 76.59 CATOM 936 C LYS A 149 −27.254 33.809 −7.319 1.00 76.35 C ATOM 937 O LYS A149 −26.822 32.672 −7.232 1.00 81.05 O ATOM 938 CB LYS A 149 −25.66235.562 −7.950 1.00 79.46 C ATOM 939 CG LYS A 149 −25.007 36.526 −8.9141.00 82.58 C ATOM 940 CD LYS A 149 −24.001 37.409 −8.129 1.00 83.85 CATOM 941 CE LYS A 149 −23.690 38.747 −8.790 1.00 89.02 C ATOM 942 NZ LYSA 149 −23.014 39.645 −7.773 1.00 88.17 N ATOM 943 N ALA A 150 −28.12034.331 −6.441 1.00 73.36 N ATOM 944 CA ALA A 150 −28.684 33.545 −5.3251.00 74.46 C ATOM 945 C ALA A 150 −29.457 32.309 −5.827 1.00 75.42 CATOM 946 O ALA A 150 −29.337 31.239 −5.245 1.00 79.11 O ATOM 947 CB ALAA 150 −29.572 34.416 −4.440 1.00 68.50 C ATOM 948 N ARG A 151 −30.24332.465 −6.900 1.00 77.55 N ATOM 949 CA ARG A 151 −30.945 31.314 −7.5111.00 77.51 C ATOM 950 C ARG A 151 −29.983 30.249 −8.022 1.00 77.61 CATOM 951 O ARG A 151 −30.272 29.057 −7.913 1.00 77.15 O ATOM 952 CB ARGA 151 −31.836 31.709 −8.690 1.00 83.00 C ATOM 953 CG ARG A 151 −33.28432.095 −8.376 1.00 89.58 C ATOM 954 CD ARG A 151 −33.576 33.556 −8.5981.00 95.56 C ATOM 955 NE ARG A 151 −34.990 33.794 −8.884 1.00 93.67 NATOM 956 CZ ARG A 151 −35.510 34.981 −9.220 1.00 98.85 C ATOM 957 NH1ARG A 151 −34.743 36.084 −9.325 1.00 97.06 N ATOM 958 NH2 ARG A 151−36.820 35.073 −9.458 1.00 102.25 N ATOM 959 N VAL A 152 −28.852 30.684−8.579 1.00 77.11 N ATOM 960 CA VAL A 152 −27.825 29.767 −9.081 1.0077.01 C ATOM 961 C VAL A 152 −27.144 29.030 −7.919 1.00 76.31 C ATOM 962O VAL A 152 −26.878 27.845 −8.023 1.00 73.81 O ATOM 963 CB VAL A 152−26.806 30.502 −9.987 1.00 78.53 C ATOM 964 CG1 VAL A 152 −25.612 29.617−10.311 1.00 85.33 C ATOM 965 CG2 VAL A 152 −27.486 30.934 −11.274 1.0075.42 C ATOM 966 N ILE A 153 −26.874 29.734 −6.824 1.00 76.01 N ATOM 967CA ILE A 153 −26.278 29.102 −5.632 1.00 76.47 C ATOM 968 C ILE A 153−27.235 28.053 −5.074 1.00 75.66 C ATOM 969 O ILE A 153 −26.812 26.945−4.733 1.00 74.99 O ATOM 970 CB ILE A 153 −25.954 30.101 −4.523 1.0075.93 C ATOM 971 CG1 ILE A 153 −24.899 31.127 −4.966 1.00 78.94 C ATOM972 CG2 ILE A 153 −25.402 29.373 −3.320 1.00 75.34 C ATOM 973 CD1 ILE A153 −23.488 30.564 −5.063 1.00 84.91 C ATOM 974 N ILE A 154 −28.51728.416 −4.990 1.00 75.41 N ATOM 975 CA ILE A 154 −29.570 27.497 −4.5451.00 75.93 C ATOM 976 C ILE A 154 −29.645 26.286 −5.461 1.00 72.61 CATOM 977 O ILE A 154 −29.565 25.159 −5.001 1.00 70.17 O ATOM 978 CB ILEA 154 −30.943 28.192 −4.490 1.00 75.92 C ATOM 979 CG1 ILE A 154 −30.97529.183 −3.328 1.00 79.46 C ATOM 980 CG2 ILE A 154 −32.072 27.170 −4.3121.00 73.88 C ATOM 981 CD1 ILE A 154 −32.130 30.133 −3.371 1.00 81.26 CATOM 982 N LEU A 155 −29.793 26.521 −6.754 1.00 75.14 N ATOM 983 CA LEUA 155 −29.826 25.411 −7.725 1.00 75.92 C ATOM 984 C LEU A 155 −28.60424.497 −7.543 1.00 75.22 C ATOM 985 O LEU A 155 −28.744 23.279 −7.5431.00 73.02 O ATOM 986 CB LEU A 155 −29.912 25.930 −9.158 1.00 71.84 CATOM 987 CG LEU A 155 −29.837 24.903 −10.297 1.00 79.20 C ATOM 988 CD1LEU A 155 −30.845 23.769 −10.095 1.00 82.92 C ATOM 989 CD2 LEU A 155−30.038 25.585 −11.666 1.00 75.89 C ATOM 990 N MET A 156 −27.423 25.100−7.384 1.00 76.89 N ATOM 991 CA MET A 156 −26.186 24.339 −7.184 1.0077.43 C ATOM 992 C MET A 156 −26.181 23.568 −5.871 1.00 76.37 C ATOM 993O MET A 156 −25.571 22.504 −5.801 1.00 78.59 O ATOM 994 CB MET A 156−24.945 25.247 −7.281 1.00 81.89 C ATOM 995 CG MET A 156 −24.609 25.744−8.716 1.00 83.04 C ATOM 996 SD MET A 156 −24.284 24.442 −9.942 1.00100.97 S ATOM 997 CE MET A 156 −24.128 25.398 −11.458 1.00 89.68 C ATOM998 N VAL A 157 −26.851 24.094 −4.841 1.00 76.00 N ATOM 999 CA VAL A 157−27.000 23.370 −3.572 1.00 74.39 C ATOM 1000 C VAL A 157 −27.835 22.098−3.784 1.00 74.09 C ATOM 1001 O VAL A 157 −27.447 21.022 −3.327 1.0074.06 O ATOM 1002 CB VAL A 157 −27.624 24.250 −2.448 1.00 73.43 C ATOM1003 CG1 VAL A 157 −28.152 23.382 −1.321 1.00 69.33 C ATOM 1004 CG2 VALA 157 −26.613 25.269 −1.942 1.00 68.63 C ATOM 1005 N TRP A 158 −28.96922.227 −4.472 1.00 74.10 N ATOM 1006 CA TRP A 158 −29.817 21.046 −4.7771.00 74.40 C ATOM 1007 C TRP A 158 −29.131 20.024 −5.683 1.00 75.32 CATOM 1008 O TRP A 158 −29.313 18.824 −5.482 1.00 78.30 O ATOM 1009 CBTRP A 158 −31.188 21.469 −5.310 1.00 70.20 C ATOM 1010 CG TRP A 158−31.961 21.950 −4.183 1.00 74.02 C ATOM 1011 CD1 TRP A 158 −32.01523.217 −3.719 1.00 80.00 C ATOM 1012 CD2 TRP A 158 −32.803 21.173 −3.3331.00 72.24 C ATOM 1013 NE1 TRP A 158 −32.842 23.290 −2.632 1.00 79.53 NATOM 1014 CE2 TRP A 158 −33.342 22.050 −2.371 1.00 79.45 C ATOM 1015 CE3TRP A 158 −33.157 19.822 −3.292 1.00 73.80 C ATOM 1016 CZ2 TRP A 158−34.220 21.625 −1.374 1.00 71.01 C ATOM 1017 CZ3 TRP A 158 −34.02619.396 −2.307 1.00 73.95 C ATOM 1018 CH2 TRP A 158 −34.549 20.299 −1.3581.00 74.98 C ATOM 1019 N ILE A 159 −28.354 20.495 −6.663 1.00 76.28 NATOM 1020 CA ILE A 159 −27.572 19.595 −7.526 1.00 75.87 C ATOM 1021 CILE A 159 −26.509 18.840 −6.713 1.00 76.85 C ATOM 1022 O ILE A 159−26.416 17.626 −6.830 1.00 76.71 O ATOM 1023 CB ILE A 159 −26.892 20.332−8.696 1.00 73.88 C ATOM 1024 CG1 ILE A 159 −27.933 20.757 −9.735 1.0075.94 C ATOM 1025 CG2 ILE A 159 −25.861 19.431 −9.354 1.00 76.05 C ATOM1026 CD1 ILE A 159 −27.359 21.529 −10.923 1.00 71.99 C ATOM 1027 N VAL A160 −25.725 19.564 −5.903 1.00 74.32 N ATOM 1028 CA VAL A 160 −24.67218.947 −5.073 1.00 74.37 C ATOM 1029 C VAL A 160 −25.257 17.970 −4.0471.00 76.12 C ATOM 1030 O VAL A 160 −24.738 16.864 −3.877 1.00 72.86 OATOM 1031 CB VAL A 160 −23.805 20.011 −4.357 1.00 75.98 C ATOM 1032 CG1VAL A 160 −22.910 19.378 −3.297 1.00 70.80 C ATOM 1033 CG2 VAL A 160−22.962 20.777 −5.377 1.00 72.65 C ATOM 1034 N SER A 161 −26.332 18.388−3.376 1.00 76.74 N ATOM 1035 CA SER A 161 −27.052 17.522 −2.436 1.0076.33 C ATOM 1036 C SER A 161 −27.588 16.253 −3.127 1.00 80.12 C ATOM1037 O SER A 161 −27.565 15.175 −2.540 1.00 81.12 O ATOM 1038 CB SER A161 −28.203 18.277 −1.783 1.00 77.19 C ATOM 1039 OG SER A 161 −27.72119.391 −1.043 1.00 76.25 O ATOM 1040 N GLY A 162 −28.067 16.399 −4.3671.00 78.97 N ATOM 1041 CA GLY A 162 −28.559 15.268 −5.165 1.00 77.45 CATOM 1042 C GLY A 162 −27.418 14.346 −5.559 1.00 75.17 C ATOM 1043 O GLYA 162 −27.536 13.134 −5.495 1.00 72.75 O ATOM 1044 N LEU A 163 −26.31314.958 −5.965 1.00 76.45 N ATOM 1045 CA LEU A 163 −25.098 14.253 −6.3551.00 78.44 C ATOM 1046 C LEU A 163 −24.517 13.473 −5.171 1.00 79.50 CATOM 1047 O LEU A 163 −23.962 12.407 −5.356 1.00 80.46 O ATOM 1048 CBLEU A 163 −24.044 15.248 −6.908 1.00 79.15 C ATOM 1049 CG LEU A 163−23.153 14.848 −8.081 1.00 79.51 C ATOM 1050 CD1 LEU A 163 −23.97814.520 −9.308 1.00 75.74 C ATOM 1051 CD2 LEU A 163 −22.183 15.984 −8.3861.00 78.31 C ATOM 1052 N THR A 164 −24.650 14.007 −3.958 1.00 82.19 NATOM 1053 CA THR A 164 −24.150 13.328 −2.751 1.00 84.31 C ATOM 1054 CTHR A 164 −25.196 12.399 −2.061 1.00 85.47 C ATOM 1055 O THR A 164−24.814 11.585 −1.225 1.00 83.60 O ATOM 1056 CB THR A 164 −23.560 14.350−1.758 1.00 84.63 C ATOM 1057 OG1 THR A 164 −24.500 15.405 −1.514 1.0086.96 O ATOM 1058 CG2 THR A 164 −22.291 14.950 −2.337 1.00 82.27 C ATOM1059 N SER A 165 −26.485 12.525 −2.415 1.00 87.63 N ATOM 1060 CA SER A165 −27.562 11.705 −1.832 1.00 88.01 C ATOM 1061 C SER A 165 −28.05710.621 −2.778 1.00 90.10 C ATOM 1062 O SER A 165 −28.029 9.438 −2.4321.00 94.95 O ATOM 1063 CB SER A 165 −28.745 12.583 −1.454 1.00 92.00 CATOM 1064 OG SER A 165 −29.324 13.166 −2.614 1.00 96.93 O ATOM 1065 NPHE A 166 −28.512 11.027 −3.967 1.00 87.60 N ATOM 1066 CA PHE A 166−29.078 10.078 −4.951 1.00 85.74 C ATOM 1067 C PHE A 166 −28.042 9.150−5.573 1.00 85.31 C ATOM 1068 O PHE A 166 −28.313 7.967 −5.704 1.0085.33 O ATOM 1069 CB PHE A 166 −29.884 10.789 −6.073 1.00 85.68 C ATOM1070 CG PHE A 166 −31.350 10.954 −5.753 1.00 84.00 C ATOM 1071 CD1 PHE A166 −32.307 10.148 −6.362 1.00 82.71 C ATOM 1072 CD2 PHE A 166 −31.77311.910 −4.847 1.00 78.83 C ATOM 1073 CE1 PHE A 166 −33.659 10.302 −6.0661.00 84.17 C ATOM 1074 CE2 PHE A 166 −33.124 12.065 −4.551 1.00 79.25 CATOM 1075 CZ PHE A 166 −34.062 11.265 −5.155 1.00 80.39 C ATOM 1076 NLEU A 167 −26.870 9.673 −5.954 1.00 83.84 N ATOM 1077 CA LEU A 167−25.839 8.813 −6.579 1.00 83.08 C ATOM 1078 C LEU A 167 −25.510 7.568−5.738 1.00 83.34 C ATOM 1079 O LEU A 167 −25.725 6.477 −6.235 1.0084.70 O ATOM 1080 CB LEU A 167 −24.543 9.568 −6.947 1.00 82.52 C ATOM1081 CG LEU A 167 −24.431 10.342 −8.250 1.00 81.28 C ATOM 1082 CD1 LEU A167 −23.003 10.847 −8.415 1.00 76.32 C ATOM 1083 CD2 LEU A 167 −24.8159.492 −9.427 1.00 75.15 C ATOM 1084 N PRO A 168 −25.003 7.727 −4.4801.00 82.18 N ATOM 1085 CA PRO A 168 −24.657 6.540 −3.663 1.00 81.22 CATOM 1086 C PRO A 168 −25.803 5.561 −3.419 1.00 80.15 C ATOM 1087 O PROA 168 −25.578 4.352 −3.439 1.00 79.49 O ATOM 1088 CB PRO A 168 −24.2267.145 −2.321 1.00 80.06 C ATOM 1089 CG PRO A 168 −23.858 8.515 −2.6141.00 82.53 C ATOM 1090 CD PRO A 168 −24.720 8.968 −3.734 1.00 83.35 CATOM 1091 N ILE A 169 −27.007 6.086 −3.191 1.00 80.34 N ATOM 1092 CA ILEA 169 −28.181 5.246 −2.931 1.00 81.83 C ATOM 1093 C ILE A 169 −28.6704.542 −4.207 1.00 83.09 C ATOM 1094 O ILE A 169 −28.974 3.347 −4.1671.00 85.69 O ATOM 1095 CB ILE A 169 −29.316 6.029 −2.245 1.00 80.40 CATOM 1096 CG1 ILE A 169 −28.845 6.522 −0.876 1.00 82.52 C ATOM 1097 CG2ILE A 169 −30.538 5.150 −2.069 1.00 80.48 C ATOM 1098 CD1 ILE A 169−29.913 7.192 −0.059 1.00 83.50 C ATOM 1099 N GLN A 170 −28.743 5.274−5.322 1.00 83.76 N ATOM 1100 CA GLN A 170 −29.161 4.691 −6.617 1.0083.63 C ATOM 1101 C GLN A 170 −28.056 3.817 −7.243 1.00 83.39 C ATOM1102 O GLN A 170 −28.374 2.864 −7.960 1.00 86.11 O ATOM 1103 CB GLN A170 −29.606 5.780 −7.616 1.00 84.12 C ATOM 1104 CG GLN A 170 −30.7666.740 −7.123 1.00 84.07 C ATOM 1105 CD GLN A 170 −32.159 6.134 −7.1191.00 80.98 C ATOM 1106 OE1 GLN A 170 −32.370 5.018 −7.557 1.00 77.62 OATOM 1107 NE2 GLN A 170 −33.123 6.895 −6.614 1.00 76.26 N ATOM 1108 NMET A 171 −26.777 4.134 −6.981 1.00 82.82 N ATOM 1109 CA MET A 171−25.636 3.286 −7.436 1.00 83.78 C ATOM 1110 C MET A 171 −25.340 2.113−6.493 1.00 83.40 C ATOM 1111 O MET A 171 −24.487 1.279 −6.816 1.0079.60 O ATOM 1112 CB MET A 171 −24.336 4.104 −7.631 1.00 84.08 C ATOM1113 CG MET A 171 −24.315 5.014 −8.854 1.00 84.82 C ATOM 1114 SD MET A171 −24.372 4.141 −10.445 1.00 93.60 S ATOM 1115 CE MET A 171 −22.9623.018 −10.356 1.00 86.88 C ATOM 1116 N HIS A 172 −26.032 2.056 −5.3461.00 83.18 N ATOM 1117 CA HIS A 172 −25.909 0.954 −4.373 1.00 83.41 CATOM 1118 C HIS A 172 −24.544 0.943 −3.621 1.00 83.06 C ATOM 1119 O HISA 172 −24.082 −0.107 −3.168 1.00 83.35 O ATOM 1120 CB HIS A 172 −26.218−0.415 −5.050 1.00 82.96 C ATOM 1121 CG HIS A 172 −27.640 −0.563 −5.5051.00 83.78 C ATOM 1122 ND1 HIS A 172 −28.273 −1.780 −5.517 1.00 79.73 NATOM 1123 CD2 HIS A 172 −28.552 0.332 −5.957 1.00 82.46 C ATOM 1124 CE1HIS A 172 −29.509 −1.639 −5.954 1.00 77.50 C ATOM 1125 NE2 HIS A 172−29.705 −0.364 −6.229 1.00 81.47 N ATOM 1126 N TRP A 173 −23.915 2.116−3.495 1.00 83.44 N ATOM 1127 CA TRP A 173 −22.647 2.253 −2.758 1.0083.86 C ATOM 1128 C TRP A 173 −22.823 2.178 −1.244 1.00 83.60 C ATOM1129 O TRP A 173 −21.888 1.833 −0.520 1.00 83.83 O ATOM 1130 CB TRP A173 −21.953 3.576 −3.084 1.00 83.86 C ATOM 1131 CG TRP A 173 −21.4923.720 −4.491 1.00 84.03 C ATOM 1132 CD1 TRP A 173 −21.496 2.766 −5.4751.00 84.91 C ATOM 1133 CD2 TRP A 173 −20.939 4.893 −5.079 1.00 82.79 CATOM 1134 NE1 TRP A 173 −20.988 3.281 −6.631 1.00 84.59 N ATOM 1135 CE2TRP A 173 −20.636 4.585 −6.419 1.00 81.34 C ATOM 1136 CE3 TRP A 173−20.671 6.184 −4.602 1.00 84.75 C ATOM 1137 CZ2 TRP A 173 −20.079 5.517−7.291 1.00 85.31 C ATOM 1138 CZ3 TRP A 173 −20.115 7.115 −5.470 1.0084.09 C ATOM 1139 CH2 TRP A 173 −19.825 6.777 −6.799 1.00 86.15 C ATOM1140 N TYR A 174 −24.018 2.505 −0.777 1.00 81.87 N ATOM 1141 CA TYR A174 −24.330 2.478 0.643 1.00 82.95 C ATOM 1142 C TYR A 174 −24.461 1.1031.261 1.00 84.39 C ATOM 1143 O TYR A 174 −24.328 0.993 2.451 1.00 85.16O ATOM 1144 CB TYR A 174 −25.646 3.224 0.916 1.00 82.89 C ATOM 1145 CGTYR A 174 −26.913 2.473 0.522 1.00 80.98 C ATOM 1146 CD1 TYR A 174−27.679 1.794 1.475 1.00 79.89 C ATOM 1147 CD2 TYR A 174 −27.340 2.442−0.798 1.00 79.78 C ATOM 1148 CE1 TYR A 174 −28.838 1.110 1.111 1.0081.50 C ATOM 1149 CE2 TYR A 174 −28.489 1.767 −1.174 1.00 80.48 C ATOM1150 CZ TYR A 174 −29.234 1.104 −0.221 1.00 81.26 C ATOM 1151 OH TYR A174 −30.365 0.439 −0.601 1.00 79.71 O ATOM 1152 N ARG A 175 −24.7090.070 0.457 1.00 82.91 N ATOM 1153 CA ARG A 175 −25.158 −1.227 0.9841.00 81.71 C ATOM 1154 C ARG A 175 −24.093 −2.047 1.678 1.00 80.96 CATOM 1155 O ARG A 175 −22.922 −1.998 1.313 1.00 80.38 O ATOM 1156 CB ARGA 175 −25.819 −2.057 −0.136 1.00 82.53 C ATOM 1157 CG ARG A 175 −26.861−1.242 −0.863 1.00 85.26 C ATOM 1158 CD ARG A 175 −28.068 −1.948 −1.3181.00 83.28 C ATOM 1159 NE ARG A 175 −27.867 −2.850 −2.413 1.00 82.86 NATOM 1160 CZ ARG A 175 −28.850 −3.346 −3.172 1.00 86.74 C ATOM 1161 NH1ARG A 175 −30.142 −3.019 −2.963 1.00 84.23 N ATOM 1162 NH2 ARG A 175−28.541 −4.184 −4.169 1.00 81.76 N ATOM 1163 N ALA A 176 −24.539 −2.7932.689 1.00 81.02 N ATOM 1164 CA ALA A 176 −23.696 −3.687 3.454 1.0081.16 C ATOM 1165 C ALA A 176 −23.664 −5.020 2.737 1.00 81.72 C ATOM1166 O ALA A 176 −24.541 −5.308 1.920 1.00 81.33 O ATOM 1167 CB ALA A176 −24.231 −3.853 4.854 1.00 81.01 C ATOM 1168 N THR A 177 −22.651−5.821 3.056 1.00 81.43 N ATOM 1169 CA THR A 177 −22.421 −7.128 2.4221.00 80.81 C ATOM 1170 C THR A 177 −22.741 −8.348 3.319 1.00 81.25 CATOM 1171 O THR A 177 −22.430 −9.481 2.947 1.00 81.14 O ATOM 1172 CB THRA 177 −20.959 −7.215 1.944 1.00 80.88 C ATOM 1173 OG1 THR A 177 −20.080−7.111 3.071 1.00 80.59 O ATOM 1174 CG2 THR A 177 −20.656 −6.090 0.9611.00 80.78 C ATOM 1175 N HIS A 178 −23.358 −8.123 4.482 1.00 80.75 NATOM 1176 CA HIS A 178 −23.719 −9.227 5.397 1.00 80.59 C ATOM 1177 C HISA 178 −25.129 −9.723 5.047 1.00 79.62 C ATOM 1178 O HIS A 178 −25.924−8.985 4.473 1.00 80.66 O ATOM 1179 CB HIS A 178 −23.574 −8.824 6.8781.00 80.59 C ATOM 1180 CG HIS A 178 −24.512 −7.752 7.320 1.00 80.24 CATOM 1181 ND1 HIS A 178 −24.229 −6.410 7.189 1.00 85.34 N ATOM 1182 CD2HIS A 178 −25.731 −7.825 7.895 1.00 81.58 C ATOM 1183 CE1 HIS A 178−25.238 −5.702 7.664 1.00 86.20 C ATOM 1184 NE2 HIS A 178 −26.163 −6.5378.098 1.00 88.13 N ATOM 1185 N GLN A 179 −25.413 −10.973 5.405 1.0079.51 N ATOM 1186 CA GLN A 179 −26.652 −11.670 4.994 1.00 79.05 C ATOM1187 C GLN A 179 −27.968 −11.024 5.453 1.00 77.54 C ATOM 1188 O GLN A179 −28.903 −10.937 4.667 1.00 76.07 O ATOM 1189 CB GLN A 179 −26.624−13.140 5.467 1.00 79.94 C ATOM 1190 CG GLN A 179 −27.540 −14.079 4.6681.00 82.48 C ATOM 1191 CD GLN A 179 −27.127 −14.218 3.201 1.00 85.58 CATOM 1192 OE1 GLN A 179 −25.963 −14.006 2.838 1.00 84.07 O ATOM 1193 NE2GLN A 179 −28.082 −14.578 2.355 1.00 88.48 N ATOM 1194 N GLU A 180−28.026 −10.587 6.713 1.00 76.40 N ATOM 1195 CA GLU A 180 −29.224 −9.9157.278 1.00 76.08 C ATOM 1196 C GLU A 180 −29.656 −8.694 6.441 1.00 74.97C ATOM 1197 O GLU A 180 −30.854 −8.437 6.268 1.00 72.77 O ATOM 1198 CBGLU A 180 −28.957 −9.518 8.738 1.00 77.18 C ATOM 1199 CG GLU A 180−30.098 −8.829 9.499 1.00 78.62 C ATOM 1200 CD GLU A 180 −29.685 −8.43010.920 1.00 78.93 C ATOM 1201 OE1 GLU A 180 −30.024 −7.303 11.336 1.0077.22 O ATOM 1202 OE2 GLU A 180 −29.022 −9.239 11.618 1.00 82.04 O ATOM1203 N ALA A 181 −28.666 −7.962 5.929 1.00 73.84 N ATOM 1204 CA ALA A181 −28.887 −6.814 5.061 1.00 74.58 C ATOM 1205 C ALA A 181 −29.359−7.291 3.699 1.00 74.81 C ATOM 1206 O ALA A 181 −30.389 −6.828 3.2011.00 74.62 O ATOM 1207 CB ALA A 181 −27.610 −6.005 4.921 1.00 72.34 CATOM 1208 N ILE A 182 −28.592 −8.218 3.110 1.00 76.12 N ATOM 1209 CA ILEA 182 −28.905 −8.843 1.800 1.00 76.87 C ATOM 1210 C ILE A 182 −30.356−9.336 1.743 1.00 77.84 C ATOM 1211 O ILE A 182 −31.047 −9.109 0.7451.00 78.53 O ATOM 1212 CB ILE A 182 −27.924 −10.003 1.453 1.00 76.51 CATOM 1213 CG1 ILE A 182 −26.515 −9.466 1.192 1.00 75.02 C ATOM 1214 CG2ILE A 182 −28.387 −10.756 0.226 1.00 76.30 C ATOM 1215 CD1 ILE A 182−25.478 −10.540 0.959 1.00 75.60 C ATOM 1216 N ASN A 183 −30.796 −10.0002.817 1.00 79.14 N ATOM 1217 CA ASN A 183 −32.185 −10.442 2.958 1.0079.97 C ATOM 1218 C ASN A 183 −33.152 −9.263 2.924 1.00 81.18 C ATOM1219 O ASN A 183 −34.160 −9.323 2.232 1.00 81.85 O ATOM 1220 CB ASN A183 −32.392 −11.225 4.260 1.00 79.70 C ATOM 1221 CG ASN A 183 −31.725−12.586 4.246 1.00 78.69 C ATOM 1222 OD1 ASN A 183 −30.711 −12.800 3.5861.00 76.53 O ATOM 1223 ND2 ASN A 183 −32.301 −13.522 4.984 1.00 78.92 NATOM 1224 N CYS A 184 −32.835 −8.200 3.671 1.00 81.90 N ATOM 1225 CA CYSA 184 −33.675 −6.990 3.698 1.00 81.53 C ATOM 1226 C CYS A 184 −33.742−6.278 2.348 1.00 80.40 C ATOM 1227 O CYS A 184 −34.791 −5.743 1.9821.00 80.62 O ATOM 1228 CB CYS A 184 −33.189 −5.995 4.751 1.00 82.03 CATOM 1229 SG CYS A 184 −34.281 −4.556 4.896 1.00 86.09 S ATOM 1230 N TYRA 185 −32.630 −6.270 1.614 1.00 80.51 N ATOM 1231 CA TYR A 185 −32.588−5.626 0.289 1.00 80.30 C ATOM 1232 C TYR A 185 −33.500 −6.328 −0.6931.00 80.59 C ATOM 1233 O TYR A 185 −34.322 −5.683 −1.354 1.00 82.14 OATOM 1234 CB TYR A 185 −31.165 −5.597 −0.278 1.00 79.80 C ATOM 1235 CGTYR A 185 −30.177 −4.798 0.552 1.00 80.24 C ATOM 1236 CD1 TYR A 185−30.497 −3.522 1.018 1.00 76.86 C ATOM 1237 CD2 TYR A 185 −28.923 −5.3130.866 1.00 79.73 C ATOM 1238 CE1 TYR A 185 −29.604 −2.797 1.769 1.0078.10 C ATOM 1239 CE2 TYR A 185 −28.021 −4.589 1.618 1.00 79.03 C ATOM1240 CZ TYR A 185 −28.365 −3.334 2.066 1.00 78.33 C ATOM 1241 OH TYR A185 −27.471 −2.621 2.811 1.00 79.23 O ATOM 1242 N ALA A 186 −33.344−7.645 −0.776 1.00 79.03 N ATOM 1243 CA ALA A 186 −34.189 −8.489 −1.6221.00 78.43 C ATOM 1244 C ALA A 186 −35.659 −8.474 −1.178 1.00 78.71 CATOM 1245 O ALA A 186 −36.557 −8.506 −2.023 1.00 79.49 O ATOM 1246 CBALA A 186 −33.661 −9.914 −1.627 1.00 76.05 C ATOM 1247 N GLU A 187−35.887 −8.422 0.140 1.00 79.25 N ATOM 1248 CA GLU A 187 −37.237 −8.3990.713 1.00 79.62 C ATOM 1249 C GLU A 187 −37.976 −7.085 0.400 1.00 79.69C ATOM 1250 O GLU A 187 −37.549 −5.998 0.814 1.00 80.47 O ATOM 1251 CBGLU A 187 −37.181 −8.643 2.234 1.00 79.95 C ATOM 1252 CG GLU A 187−38.523 −8.617 2.992 1.00 82.26 C ATOM 1253 CD GLU A 187 −39.521 −9.6712.533 1.00 86.24 C ATOM 1254 OE1 GLU A 187 −39.137 −10.643 1.845 1.0092.89 O ATOM 1255 OE2 GLU A 187 −40.711 −9.530 2.863 1.00 88.95 O ATOM1256 N GLU A 188 −39.081 −7.216 −0.334 1.00 79.07 N ATOM 1257 CA GLU A188 −39.930 −6.085 −0.751 1.00 79.55 C ATOM 1258 C GLU A 188 −40.619−5.361 0.404 1.00 79.89 C ATOM 1259 O GLU A 188 −40.897 −4.161 0.2921.00 79.15 O ATOM 1260 CB GLU A 188 −40.983 −6.562 −1.747 1.00 79.62 CATOM 1261 CG GLU A 188 −42.048 −7.458 −1.121 1.00 83.66 C ATOM 1262 CDGLU A 188 −42.614 −8.463 −2.084 1.00 84.99 C ATOM 1263 OE1 GLU A 188−42.812 −8.122 −3.268 1.00 95.21 O ATOM 1264 OE2 GLU A 188 −42.866−9.605 −1.652 1.00 89.59 O ATOM 1265 N THR A 189 −40.891 −6.089 1.4961.00 77.96 N ATOM 1266 CA THR A 189 −41.533 −5.512 2.676 1.00 75.91 CATOM 1267 C THR A 189 −40.541 −5.063 3.755 1.00 76.43 C ATOM 1268 O THRA 189 −40.961 −4.656 4.836 1.00 76.97 O ATOM 1269 CB THR A 189 −42.562−6.481 3.300 1.00 75.78 C ATOM 1270 OG1 THR A 189 −41.883 −7.617 3.8431.00 74.95 O ATOM 1271 CG2 THR A 189 −43.580 −6.923 2.252 1.00 70.98 CATOM 1272 N CYS A 190 −39.242 −5.131 3.464 1.00 78.00 N ATOM 1273 CA CYSA 190 −38.209 −4.690 4.377 1.00 77.37 C ATOM 1274 C CYS A 190 −37.588−3.443 3.799 1.00 74.95 C ATOM 1275 O CYS A 190 −37.131 −3.464 2.6581.00 75.26 O ATOM 1276 CB CYS A 190 −37.136 −5.755 4.545 1.00 79.47 CATOM 1277 SG CYS A 190 −35.924 −5.347 5.847 1.00 85.35 S ATOM 1278 N CYSA 191 −37.574 −2.368 4.582 1.00 75.05 N ATOM 1279 CA CYS A 191 −36.955−1.110 4.175 1.00 77.17 C ATOM 1280 C CYS A 191 −36.140 −0.537 5.3291.00 77.29 C ATOM 1281 O CYS A 191 −36.450 0.523 5.856 1.00 81.42 O ATOM1282 CB CYS A 191 −38.024 −0.135 3.680 1.00 77.85 C ATOM 1283 SG CYS A191 −37.383 1.370 2.899 1.00 80.61 S ATOM 1284 N ASP A 192 −35.091−1.270 5.703 1.00 78.03 N ATOM 1285 CA ASP A 192 −34.141 −0.851 6.7411.00 76.97 C ATOM 1286 C ASP A 192 −32.909 −0.347 6.054 1.00 75.94 CATOM 1287 O ASP A 192 −32.428 −0.979 5.114 1.00 77.63 O ATOM 1288 CB ASPA 192 −33.762 −2.005 7.667 1.00 77.44 C ATOM 1289 CG ASP A 192 −34.889−2.419 8.586 1.00 80.00 C ATOM 1290 OD1 ASP A 192 −35.724 −1.561 8.9591.00 82.18 O ATOM 1291 OD2 ASP A 192 −34.941 −3.619 8.943 1.00 80.09 OATOM 1292 N PHE A 193 −32.388 0.785 6.517 1.00 75.48 N ATOM 1293 CA PHEA 193 −31.237 1.416 5.890 1.00 75.22 C ATOM 1294 C PHE A 193 −29.9090.865 6.443 1.00 75.43 C ATOM 1295 O PHE A 193 −29.160 1.573 7.133 1.0073.54 O ATOM 1296 CB PHE A 193 −31.351 2.947 6.043 1.00 74.11 C ATOM1297 CG PHE A 193 −30.399 3.730 5.175 1.00 76.00 C ATOM 1298 CD1 PHE A193 −30.300 3.471 3.793 1.00 76.25 C ATOM 1299 CD2 PHE A 193 −29.6004.736 5.724 1.00 76.96 C ATOM 1300 CE1 PHE A 193 −29.428 4.189 2.9841.00 73.80 C ATOM 1301 CE2 PHE A 193 −28.718 5.465 4.905 1.00 79.72 CATOM 1302 CZ PHE A 193 −28.638 5.183 3.528 1.00 75.51 C ATOM 1303 N PHEA 194 −29.641 −0.409 6.127 1.00 74.51 N ATOM 1304 CA PHE A 194 −28.376−1.061 6.455 1.00 74.06 C ATOM 1305 C PHE A 194 −27.340 −0.493 5.5271.00 74.74 C ATOM 1306 O PHE A 194 −27.542 −0.522 4.308 1.00 73.83 OATOM 1307 CB PHE A 194 −28.423 −2.577 6.235 1.00 73.04 C ATOM 1308 CGPHE A 194 −29.226 −3.318 7.256 1.00 73.26 C ATOM 1309 CD1 PHE A 194−28.674 −3.630 8.499 1.00 75.81 C ATOM 1310 CD2 PHE A 194 −30.534 −3.7126.984 1.00 74.89 C ATOM 1311 CE1 PHE A 194 −29.412 −4.320 9.457 1.0074.15 C ATOM 1312 CE2 PHE A 194 −31.282 −4.405 7.936 1.00 75.40 C ATOM1313 CZ PHE A 194 −30.720 −4.709 9.176 1.00 74.86 C ATOM 1314 N THR A195 −26.241 0.022 6.087 1.00 73.91 N ATOM 1315 CA THR A 195 −25.1570.559 5.278 1.00 72.89 C ATOM 1316 C THR A 195 −23.811 −0.039 5.644 1.0071.51 C ATOM 1317 O THR A 195 −23.636 −0.565 6.748 1.00 70.29 O ATOM1318 CB THR A 195 −25.034 2.092 5.403 1.00 73.36 C ATOM 1319 OG1 THR A195 −24.316 2.431 6.598 1.00 75.79 O ATOM 1320 CG2 THR A 195 −26.4152.762 5.409 1.00 75.39 C ATOM 1321 N ASN A 196 −22.863 0.047 4.709 1.0069.76 N ATOM 1322 CA ASN A 196 −21.483 −0.332 4.996 1.00 70.25 C ATOM1323 C ASN A 196 −20.870 0.823 5.783 1.00 70.56 C ATOM 1324 O ASN A 196−21.350 1.967 5.694 1.00 73.34 O ATOM 1325 CB ASN A 196 −20.676 −0.7213.732 1.00 70.71 C ATOM 1326 CG ASN A 196 −20.478 0.426 2.743 1.00 71.43C ATOM 1327 OD1 ASN A 196 −19.940 1.481 3.071 1.00 74.37 O ATOM 1328 ND2ASN A 196 −20.920 0.205 1.515 1.00 72.46 N ATOM 1329 N GLN A 197 −19.8240.524 6.544 1.00 69.71 N ATOM 1330 CA GLN A 197 −19.195 1.507 7.446 1.0069.84 C ATOM 1331 C GLN A 197 −18.570 2.687 6.711 1.00 68.45 C ATOM 1332O GLN A 197 −18.745 3.832 7.137 1.00 68.34 O ATOM 1333 CB GLN A 197−18.141 0.829 8.319 1.00 70.49 C ATOM 1334 CG GLN A 197 −18.723 −0.2219.264 1.00 73.78 C ATOM 1335 CD GLN A 197 −17.662 −1.017 9.977 1.0075.55 C ATOM 1336 OE1 GLN A 197 −16.556 −0.528 10.223 1.00 81.19 O ATOM1337 NE2 GLN A 197 −17.988 −2.262 10.319 1.00 83.68 N ATOM 1338 N ALA A198 −17.855 2.400 5.617 1.00 67.03 N ATOM 1339 CA ALA A 198 −17.2223.430 4.772 1.00 67.06 C ATOM 1340 C ALA A 198 −18.229 4.480 4.309 1.0068.00 C ATOM 1341 O ALA A 198 −17.926 5.672 4.310 1.00 67.52 O ATOM 1342CB ALA A 198 −16.540 2.796 3.576 1.00 67.06 C ATOM 1343 N TYR A 199−19.420 4.027 3.916 1.00 67.41 N ATOM 1344 CA TYR A 199 −20.503 4.9253.544 1.00 68.01 C ATOM 1345 C TYR A 199 −21.001 5.678 4.753 1.00 66.60C ATOM 1346 O TYR A 199 −21.162 6.889 4.697 1.00 64.41 O ATOM 1347 CBTYR A 199 −21.685 4.168 2.935 1.00 69.88 C ATOM 1348 CG TYR A 199−22.897 5.054 2.714 1.00 67.04 C ATOM 1349 CD1 TYR A 199 −23.009 5.8311.566 1.00 70.50 C ATOM 1350 CD2 TYR A 199 −23.932 5.113 3.655 1.0069.18 C ATOM 1351 CE1 TYR A 199 −24.128 6.657 1.352 1.00 71.51 C ATOM1352 CE2 TYR A 199 −25.051 5.933 3.453 1.00 72.40 C ATOM 1353 CZ TYR A199 −25.141 6.702 2.298 1.00 71.80 C ATOM 1354 OH TYR A 199 −26.2367.509 2.088 1.00 73.79 O ATOM 1355 N ALA A 200 −21.245 4.942 5.840 1.0067.80 N ATOM 1356 CA ALA A 200 −21.723 5.531 7.100 1.00 66.98 C ATOM1357 C ALA A 200 −20.840 6.697 7.533 1.00 65.35 C ATOM 1358 O ALA A 200−21.354 7.739 7.890 1.00 62.59 O ATOM 1359 CB ALA A 200 −21.789 4.4868.183 1.00 67.92 C ATOM 1360 N ILE A 201 −19.520 6.505 7.489 1.00 66.75N ATOM 1361 CA ILE A 201 −18.559 7.565 7.830 1.00 69.39 C ATOM 1362 CILE A 201 −18.514 8.680 6.785 1.00 70.99 C ATOM 1363 O ILE A 201 −18.7689.836 7.107 1.00 71.46 O ATOM 1364 CB ILE A 201 −17.126 7.027 7.971 1.0068.97 C ATOM 1365 CG1 ILE A 201 −16.998 6.142 9.210 1.00 70.83 C ATOM1366 CG2 ILE A 201 −16.142 8.181 8.064 1.00 68.40 C ATOM 1367 CD1 ILE A201 −15.610 5.530 9.383 1.00 71.18 C ATOM 1368 N ALA A 202 −18.191 8.3115.542 1.00 71.53 N ATOM 1369 CA ALA A 202 −18.001 9.273 4.438 1.00 70.39C ATOM 1370 C ALA A 202 −19.228 10.112 4.136 1.00 71.21 C ATOM 1371 OALA A 202 −19.112 11.331 3.979 1.00 72.58 O ATOM 1372 CB ALA A 202−17.558 8.555 3.177 1.00 69.73 C ATOM 1373 N SER A 203 −20.395 9.4744.054 1.00 71.88 N ATOM 1374 CA SER A 203 −21.633 10.202 3.748 1.0072.73 C ATOM 1375 C SER A 203 −22.030 11.160 4.861 1.00 75.38 C ATOM1376 O SER A 203 −22.457 12.274 4.579 1.00 76.69 O ATOM 1377 CB SER A203 −22.798 9.261 3.492 1.00 73.42 C ATOM 1378 OG SER A 203 −23.94410.010 3.118 1.00 77.91 O ATOM 1379 N SER A 204 −21.890 10.730 6.1141.00 72.53 N ATOM 1380 CA SER A 204 −22.266 11.570 7.250 1.00 71.77 CATOM 1381 C SER A 204 −21.283 12.742 7.442 1.00 73.36 C ATOM 1382 O SERA 204 −21.695 13.822 7.863 1.00 73.43 O ATOM 1383 CB SER A 204 −22.42810.728 8.525 1.00 69.52 C ATOM 1384 OG SER A 204 −21.292 9.941 8.7761.00 76.74 O ATOM 1385 N ILE A 205 −19.998 12.537 7.137 1.00 73.22 NATOM 1386 CA ILE A 205 −19.020 13.638 7.213 1.00 73.31 C ATOM 1387 C ILEA 205 −19.284 14.674 6.118 1.00 76.06 C ATOM 1388 O ILE A 205 −19.39915.872 6.405 1.00 76.12 O ATOM 1389 CB ILE A 205 −17.569 13.147 7.1261.00 72.39 C ATOM 1390 CG1 ILE A 205 −17.173 12.458 8.435 1.00 71.69 CATOM 1391 CG2 ILE A 205 −16.625 14.303 6.876 1.00 70.67 C ATOM 1392 CD1ILE A 205 −15.812 11.794 8.390 1.00 72.10 C ATOM 1393 N VAL A 206−19.380 14.201 4.877 1.00 74.65 N ATOM 1394 CA VAL A 206 −19.591 15.0813.713 1.00 77.11 C ATOM 1395 C VAL A 206 −20.972 15.757 3.665 1.00 75.89C ATOM 1396 O VAL A 206 −21.071 16.933 3.317 1.00 75.71 O ATOM 1397 CBVAL A 206 −19.356 14.321 2.388 1.00 75.98 C ATOM 1398 CG1 VAL A 206−19.647 15.210 1.194 1.00 77.29 C ATOM 1399 CG2 VAL A 206 −17.920 13.8192.324 1.00 80.65 C ATOM 1400 N SER A 207 −22.023 15.025 4.009 1.00 76.66N ATOM 1401 CA SER A 207 −23.377 15.585 3.972 1.00 77.79 C ATOM 1402 CSER A 207 −23.720 16.457 5.165 1.00 78.97 C ATOM 1403 O SER A 207−24.437 17.437 4.995 1.00 83.06 O ATOM 1404 CB SER A 207 −24.420 14.4883.922 1.00 78.06 C ATOM 1405 OG SER A 207 −24.231 13.633 2.814 1.0083.89 O ATOM 1406 N PHE A 208 −23.216 16.103 6.356 1.00 79.98 N ATOM1407 CA PHE A 208 −23.601 16.771 7.609 1.00 77.07 C ATOM 1408 C PHE A208 −22.463 17.477 8.319 1.00 78.35 C ATOM 1409 O PHE A 208 −22.51118.695 8.470 1.00 79.76 O ATOM 1410 CB PHE A 208 −24.254 15.753 8.5521.00 76.98 C ATOM 1411 CG PHE A 208 −24.676 16.321 9.888 1.00 78.18 CATOM 1412 CD1 PHE A 208 −25.792 17.141 9.984 1.00 77.60 C ATOM 1413 CD2PHE A 208 −23.957 16.025 11.051 1.00 75.04 C ATOM 1414 CE1 PHE A 208−26.190 17.668 11.216 1.00 77.26 C ATOM 1415 CE2 PHE A 208 −24.35016.545 12.278 1.00 80.26 C ATOM 1416 CZ PHE A 208 −25.475 17.373 12.3571.00 77.27 C ATOM 1417 N TYR A 209 −21.450 16.739 8.756 1.00 78.29 NATOM 1418 CA TYR A 209 −20.386 17.351 9.593 1.00 78.96 C ATOM 1419 C TYRA 209 −19.583 18.463 8.941 1.00 78.89 C ATOM 1420 O TYR A 209 −19.22219.422 9.625 1.00 79.13 O ATOM 1421 CB TYR A 209 −19.425 16.295 10.1311.00 80.47 C ATOM 1422 CG TYR A 209 −20.047 15.425 11.181 1.00 82.05 CATOM 1423 CD1 TYR A 209 −20.330 14.088 10.937 1.00 80.35 C ATOM 1424 CD2TYR A 209 −20.360 15.948 12.440 1.00 84.46 C ATOM 1425 CE1 TYR A 209−20.906 13.292 11.913 1.00 83.44 C ATOM 1426 CE2 TYR A 209 −20.93615.163 13.424 1.00 85.84 C ATOM 1427 CZ TYR A 209 −21.208 13.835 13.1591.00 85.25 C ATOM 1428 OH TYR A 209 −21.776 13.073 14.145 1.00 89.70 OATOM 1429 N VAL A 210 −19.304 18.351 7.645 1.00 78.18 N ATOM 1430 CA VALA 210 −18.557 19.404 6.944 1.00 77.41 C ATOM 1431 C VAL A 210 −19.35020.740 6.944 1.00 77.38 C ATOM 1432 O VAL A 210 −18.853 21.716 7.4951.00 80.02 O ATOM 1433 CB VAL A 210 −18.108 18.976 5.515 1.00 78.51 CATOM 1434 CG1 VAL A 210 −17.758 20.202 4.653 1.00 73.94 C ATOM 1435 CG2VAL A 210 −16.932 18.022 5.609 1.00 76.43 C ATOM 1436 N PRO A 211−20.557 20.780 6.339 1.00 75.78 N ATOM 1437 CA PRO A 211 −21.304 22.0336.379 1.00 76.70 C ATOM 1438 C PRO A 211 −21.721 22.509 7.793 1.00 78.77C ATOM 1439 O PRO A 211 −21.917 23.707 7.967 1.00 81.95 O ATOM 1440 CBPRO A 211 −22.538 21.738 5.506 1.00 78.00 C ATOM 1441 CG PRO A 211−22.687 20.307 5.514 1.00 77.13 C ATOM 1442 CD PRO A 211 −21.305 19.7465.601 1.00 80.23 C ATOM 1443 N LEU A 212 −21.848 21.598 8.767 1.00 77.02N ATOM 1444 CA LEU A 212 −22.178 21.965 10.159 1.00 75.25 C ATOM 1445 CLEU A 212 −21.034 22.723 10.815 1.00 75.40 C ATOM 1446 O LEU A 212−21.248 23.765 11.404 1.00 76.71 O ATOM 1447 CB LEU A 212 −22.492 20.72711.017 1.00 73.79 C ATOM 1448 CG LEU A 212 −22.759 20.906 12.537 1.0077.60 C ATOM 1449 CD1 LEU A 212 −24.102 21.571 12.759 1.00 79.25 C ATOM1450 CD2 LEU A 212 −22.691 19.558 13.306 1.00 70.72 C ATOM 1451 N VAL A213 −19.826 22.183 10.711 1.00 76.14 N ATOM 1452 CA VAL A 213 −18.63622.808 11.311 1.00 76.70 C ATOM 1453 C VAL A 213 −18.372 24.190 10.7131.00 77.08 C ATOM 1454 O VAL A 213 −18.039 25.132 11.437 1.00 76.69 OATOM 1455 CB VAL A 213 −17.380 21.915 11.146 1.00 79.00 C ATOM 1456 CG1VAL A 213 −16.111 22.656 11.562 1.00 78.68 C ATOM 1457 CG2 VAL A 213−17.534 20.627 11.955 1.00 77.70 C ATOM 1458 N ILE A 214 −18.528 24.2959.396 1.00 74.65 N ATOM 1459 CA ILE A 214 −18.372 25.561 8.690 1.0074.83 C ATOM 1460 C ILE A 214 −19.423 26.574 9.162 1.00 72.99 C ATOM1461 O ILE A 214 −19.075 27.692 9.502 1.00 72.81 O ATOM 1462 CB ILE A214 −18.480 25.384 7.144 1.00 73.17 C ATOM 1463 CG1 ILE A 214 −17.31824.547 6.603 1.00 76.80 C ATOM 1464 CG2 ILE A 214 −18.501 26.748 6.4501.00 71.35 C ATOM 1465 CD1 ILE A 214 −17.443 24.202 5.126 1.00 72.96 CATOM 1466 N MET A 215 −20.692 26.159 9.171 1.00 73.55 N ATOM 1467 CA META 215 −21.824 26.990 9.611 1.00 75.44 C ATOM 1468 C MET A 215 −21.58927.580 10.979 1.00 77.02 C ATOM 1469 O MET A 215 −21.690 28.787 11.1501.00 79.77 O ATOM 1470 CB MET A 215 −23.099 26.153 9.675 1.00 74.74 CATOM 1471 CG MET A 215 −24.374 26.874 10.124 1.00 73.35 C ATOM 1472 SDMET A 215 −25.653 25.672 10.545 1.00 82.79 S ATOM 1473 CE MET A 215−25.102 25.185 12.188 1.00 78.98 C ATOM 1474 N VAL A 216 −21.281 26.70911.938 1.00 73.57 N ATOM 1475 CA VAL A 216 −21.029 27.111 13.319 1.0077.98 C ATOM 1476 C VAL A 216 −19.847 28.077 13.424 1.00 78.25 C ATOM1477 O VAL A 216 −19.960 29.124 14.061 1.00 82.48 O ATOM 1478 CB VAL A216 −20.760 25.888 14.249 1.00 74.95 C ATOM 1479 CG1 VAL A 216 −20.35826.350 15.613 1.00 73.89 C ATOM 1480 CG2 VAL A 216 −21.995 24.992 14.3341.00 72.42 C ATOM 1481 N PHE A 217 −18.724 27.724 12.802 1.00 78.36 NATOM 1482 CA PHE A 217 −17.559 28.595 12.820 1.00 77.83 C ATOM 1483 CPHE A 217 −17.880 29.962 12.227 1.00 74.98 C ATOM 1484 O PHE A 217−17.661 30.981 12.862 1.00 74.40 O ATOM 1485 CB PHE A 217 −16.394 27.99212.054 1.00 80.26 C ATOM 1486 CG PHE A 217 −15.236 28.930 11.918 1.0083.15 C ATOM 1487 CD1 PHE A 217 −14.381 29.152 12.994 1.00 87.33 C ATOM1488 CD2 PHE A 217 −14.993 29.600 10.717 1.00 87.93 C ATOM 1489 CE1 PHEA 217 −13.296 30.029 12.881 1.00 85.31 C ATOM 1490 CE2 PHE A 217 −13.91230.478 10.592 1.00 87.26 C ATOM 1491 CZ PHE A 217 −13.063 30.692 11.6771.00 86.57 C ATOM 1492 N VAL A 218 −18.400 29.958 11.009 1.00 72.95 NATOM 1493 CA VAL A 218 −18.693 31.202 10.299 1.00 72.35 C ATOM 1494 CVAL A 218 −19.739 32.039 11.007 1.00 70.18 C ATOM 1495 O VAL A 218−19.547 33.238 11.123 1.00 72.08 O ATOM 1496 CB VAL A 218 −19.126 30.9658.828 1.00 69.97 C ATOM 1497 CG1 VAL A 218 −19.608 32.264 8.189 1.0063.51 C ATOM 1498 CG2 VAL A 218 −17.973 30.393 8.039 1.00 69.43 C ATOM1499 N TYR A 219 −20.822 31.428 11.479 1.00 69.91 N ATOM 1500 CA TYR A219 −21.867 32.204 12.154 1.00 70.79 C ATOM 1501 C TYR A 219 −21.48232.646 13.561 1.00 72.41 C ATOM 1502 O TYR A 219 −22.135 33.532 14.0941.00 73.85 O ATOM 1503 CB TYR A 219 −23.216 31.482 12.201 1.00 70.68 CATOM 1504 CG TYR A 219 −24.373 32.468 12.149 1.00 72.35 C ATOM 1505 CD1TYR A 219 −24.698 33.112 10.968 1.00 75.81 C ATOM 1506 CD2 TYR A 219−25.136 32.755 13.263 1.00 77.93 C ATOM 1507 CE1 TYR A 219 −25.75734.025 10.892 1.00 74.63 C ATOM 1508 CE2 TYR A 219 −26.203 33.673 13.1901.00 70.80 C ATOM 1509 CZ TYR A 219 −26.499 34.292 12.013 1.00 79.33 CATOM 1510 OH TYR A 219 −27.544 35.187 11.958 1.00 75.98 O ATOM 1511 NSER A 220 −20.451 32.053 14.170 1.00 71.26 N ATOM 1512 CA SER A 220−19.982 32.579 15.456 1.00 72.46 C ATOM 1513 C SER A 220 −19.265 33.88615.124 1.00 73.33 C ATOM 1514 O SER A 220 −19.384 34.833 15.876 1.0071.17 O ATOM 1515 CB SER A 220 −19.110 31.593 16.240 1.00 71.03 C ATOM1516 OG SER A 220 −17.988 31.169 15.514 1.00 78.76 O ATOM 1517 N ARG A221 −18.539 33.924 13.990 1.00 71.00 N ATOM 1518 CA ARG A 221 −17.87835.164 13.507 1.00 71.92 C ATOM 1519 C ARG A 221 −18.847 36.268 13.1021.00 71.03 C ATOM 1520 O ARG A 221 −18.485 37.440 13.144 1.00 75.74 OATOM 1521 CB ARG A 221 −16.940 34.887 12.319 1.00 73.28 C ATOM 1522 CGARG A 221 −15.789 33.967 12.607 1.00 81.79 C ATOM 1523 CD ARG A 221−14.737 34.643 13.460 1.00 91.32 C ATOM 1524 NE ARG A 221 −13.695 33.71113.900 1.00 99.40 N ATOM 1525 CZ ARG A 221 −12.642 34.037 14.669 1.00104.74 C ATOM 1526 NH1 ARG A 221 −12.463 35.296 15.107 1.00 108.58 NATOM 1527 NH2 ARG A 221 −11.754 33.095 15.009 1.00 102.27 N ATOM 1528 NVAL A 222 −20.065 35.898 12.705 1.00 72.12 N ATOM 1529 CA VAL A 222−21.114 36.862 12.390 1.00 70.08 C ATOM 1530 C VAL A 222 −21.544 37.55513.680 1.00 72.52 C ATOM 1531 O VAL A 222 −21.666 38.787 13.704 1.0074.66 O ATOM 1532 CB VAL A 222 −22.314 36.199 11.688 1.00 69.66 C ATOM1533 CG1 VAL A 222 −23.527 37.099 11.713 1.00 70.73 C ATOM 1534 CG2 VALA 222 −21.940 35.844 10.245 1.00 74.61 C ATOM 1535 N PHE A 223 −21.77136.782 14.752 1.00 72.29 N ATOM 1536 CA PHE A 223 −22.115 37.388 16.0541.00 71.42 C ATOM 1537 C PHE A 223 −21.020 38.303 16.562 1.00 69.83 CATOM 1538 O PHE A 223 −21.324 39.411 16.970 1.00 71.03 O ATOM 1539 CBPHE A 223 −22.461 36.334 17.105 1.00 72.71 C ATOM 1540 CG PHE A 223−23.766 35.640 16.857 1.00 69.91 C ATOM 1541 CD1 PHE A 223 −24.92736.370 16.563 1.00 71.96 C ATOM 1542 CD2 PHE A 223 −23.850 34.266 16.9201.00 73.93 C ATOM 1543 CE1 PHE A 223 −26.121 35.741 16.338 1.00 73.93 CATOM 1544 CE2 PHE A 223 −25.055 33.624 16.693 1.00 76.95 C ATOM 1545 CZPHE A 223 −26.190 34.367 16.404 1.00 78.80 C ATOM 1546 N GLN A 224−19.766 37.854 16.534 1.00 70.23 N ATOM 1547 CA GLN A 224 −18.632 38.72416.916 1.00 71.28 C ATOM 1548 C GLN A 224 −18.655 40.034 16.155 1.0072.34 C ATOM 1549 O GLN A 224 −18.462 41.077 16.742 1.00 76.18 O ATOM1550 CB GLN A 224 −17.293 38.077 16.639 1.00 71.04 C ATOM 1551 CG GLN A224 −17.007 36.807 17.433 1.00 82.15 C ATOM 1552 CD GLN A 224 −15.56536.308 17.276 1.00 85.53 C ATOM 1553 OE1 GLN A 224 −14.706 36.986 16.6731.00 85.57 O ATOM 1554 NE2 GLN A 224 −15.290 35.107 17.824 1.00 87.83 NATOM 1555 N GLU A 225 −18.892 39.969 14.839 1.00 74.23 N ATOM 1556 CAGLU A 225 −18.986 41.187 14.026 1.00 73.90 C ATOM 1557 C GLU A 225−20.161 42.043 14.442 1.00 72.00 C ATOM 1558 O GLU A 225 −19.987 43.22114.670 1.00 69.39 O ATOM 1559 CB GLU A 225 −19.073 40.880 12.525 1.0075.78 C ATOM 1560 CG GLU A 225 −17.713 40.806 11.881 1.00 85.48 C ATOM1561 CD GLU A 225 −16.990 42.134 11.934 1.00 92.43 C ATOM 1562 OE1 GLU A225 −17.670 43.174 11.759 1.00 86.05 O ATOM 1563 OE2 GLU A 225 −15.75542.133 12.152 1.00 98.53 O ATOM 1564 N ALA A 226 −21.336 41.439 14.5371.00 70.43 N ATOM 1565 CA ALA A 226 −22.521 42.145 14.988 1.00 71.14 CATOM 1566 C ALA A 226 −22.238 42.861 16.326 1.00 70.13 C ATOM 1567 O ALAA 226 −22.513 44.040 16.455 1.00 69.11 O ATOM 1568 CB ALA A 226 −23.68341.195 15.114 1.00 69.59 C ATOM 1569 N LYS A 227 −21.686 42.139 17.2981.00 70.92 N ATOM 1570 CA LYS A 227 −21.345 42.716 18.619 1.00 72.35 CATOM 1571 C LYS A 227 −20.374 43.880 18.510 1.00 70.68 C ATOM 1572 O LYSA 227 −20.498 44.860 19.238 1.00 71.56 O ATOM 1573 CB LYS A 227 −20.72841.660 19.540 1.00 72.29 C ATOM 1574 CG LYS A 227 −20.673 42.077 21.0271.00 77.11 C ATOM 1575 CD LYS A 227 −20.272 40.913 21.948 1.00 79.33 CATOM 1576 CE LYS A 227 −20.366 41.295 23.457 1.00 89.68 C ATOM 1577 NZLYS A 227 −19.211 42.084 23.975 1.00 91.63 N ATOM 1578 N ARG A 228−19.419 43.749 17.591 1.00 71.06 N ATOM 1579 CA ARG A 228 −18.380 44.74917.349 1.00 71.67 C ATOM 1580 C ARG A 228 −18.909 46.028 16.696 1.0066.45 C ATOM 1581 O ARG A 228 −18.234 47.031 16.714 1.00 70.70 O ATOM1582 CB ARG A 228 −17.271 44.142 16.486 1.00 73.73 C ATOM 1583 CG ARG A228 −15.959 44.946 16.395 1.00 80.33 C ATOM 1584 CD ARG A 228 −15.06044.487 15.239 1.00 85.20 C ATOM 1585 NE ARG A 228 −15.691 44.699 13.9311.00 95.73 N ATOM 1586 CZ ARG A 228 −15.736 45.853 13.239 1.00 99.68 CATOM 1587 NH1 ARG A 228 −15.178 46.979 13.702 1.00 98.15 N ATOM 1588 NH2ARG A 228 −16.353 45.889 12.052 1.00 97.65 N ATOM 1589 N GLN A 229−20.105 46.002 16.127 1.00 67.44 N ATOM 1590 CA GLN A 229 −20.718 47.21715.562 1.00 69.07 C ATOM 1591 C GLN A 229 −21.324 48.107 16.630 1.0068.99 C ATOM 1592 O GLN A 229 −21.550 49.299 16.388 1.00 73.07 O ATOM1593 CB GLN A 229 −21.817 46.858 14.603 1.00 67.27 C ATOM 1594 CG GLN A229 −21.328 46.085 13.403 1.00 79.11 C ATOM 1595 CD GLN A 229 −22.41245.836 12.358 1.00 81.84 C ATOM 1596 OE1 GLN A 229 −22.097 45.437 11.2331.00 98.22 O ATOM 1597 NE2 GLN A 229 −23.703 46.069 12.724 1.00 80.39 NATOM 1598 N LEU A 230 −21.594 47.546 17.804 1.00 65.76 N ATOM 1599 CALEU A 230 −22.180 48.318 18.887 1.00 66.37 C ATOM 1600 C LEU A 230−21.228 49.424 19.268 1.00 62.49 C ATOM 1601 O LEU A 230 −20.068 49.18419.517 1.00 63.24 O ATOM 1602 CB LEU A 230 −22.492 47.429 20.078 1.0063.29 C ATOM 1603 CG LEU A 230 −23.526 46.346 19.805 1.00 63.55 C ATOM1604 CD1 LEU A 230 −23.659 45.401 21.017 1.00 56.14 C ATOM 1605 CD2 LEUA 230 −24.867 46.998 19.398 1.00 63.01 C ATOM 1606 N ASN A 1002 −21.74550.645 19.298 1.00 65.96 N ATOM 1607 CA ASN A 1002 −20.959 51.846 19.6321.00 68.64 C ATOM 1608 C ASN A 1002 −21.911 52.946 20.059 1.00 68.56 CATOM 1609 O ASN A 1002 −23.125 52.706 20.134 1.00 70.46 O ATOM 1610 CBASN A 1002 −20.114 52.260 18.442 1.00 68.39 C ATOM 1611 CG ASN A 1002−20.935 52.517 17.224 1.00 74.98 C ATOM 1612 OD1 ASN A 1002 −22.10052.934 17.313 1.00 65.74 O ATOM 1613 ND2 ASN A 1002 −20.342 52.27316.058 1.00 67.18 N ATOM 1614 N ILE A 1003 −21.390 54.139 20.330 1.0069.49 N ATOM 1615 CA ILE A 1003 −22.224 55.203 20.901 1.00 70.87 C ATOM1616 C ILE A 1003 −23.404 55.572 20.013 1.00 70.78 C ATOM 1617 O ILE A1003 −24.472 55.886 20.521 1.00 74.04 O ATOM 1618 CB ILE A 1003 −21.36256.453 21.310 1.00 74.80 C ATOM 1619 CG1 ILE A 1003 −22.172 57.39022.229 1.00 76.63 C ATOM 1620 CG2 ILE A 1003 −20.788 57.153 20.075 1.0068.47 C ATOM 1621 CD1 ILE A 1003 −21.370 58.560 22.737 1.00 73.42 C ATOM1622 N PHE A 1004 −23.204 55.527 18.695 1.00 73.63 N ATOM 1623 CA PHE A1004 −24.261 55.801 17.719 1.00 72.72 C ATOM 1624 C PHE A 1004 −25.40954.841 17.835 1.00 73.43 C ATOM 1625 O PHE A 1004 −26.574 55.255 17.8721.00 71.77 O ATOM 1626 CB PHE A 1004 −23.679 55.727 16.309 1.00 75.62 CATOM 1627 CG PHE A 1004 −24.689 55.716 15.236 1.00 71.84 C ATOM 1628 CD1PHE A 1004 −25.453 56.848 14.979 1.00 82.28 C ATOM 1629 CD2 PHE A 1004−24.892 54.570 14.452 1.00 82.90 C ATOM 1630 CE1 PHE A 1004 −26.42356.843 13.943 1.00 77.44 C ATOM 1631 CE2 PHE A 1004 −25.865 54.56413.410 1.00 71.41 C ATOM 1632 CZ PHE A 1004 −26.620 55.697 13.166 1.0070.16 C ATOM 1633 N GLU A 1005 −25.074 53.559 17.889 1.00 72.98 N ATOM1634 CA GLU A 1005 −26.063 52.517 18.064 1.00 72.41 C ATOM 1635 C GLU A1005 −26.733 52.619 19.416 1.00 74.97 C ATOM 1636 O GLU A 1005 −27.94952.434 19.517 1.00 74.19 O ATOM 1637 CB GLU A 1005 −25.418 51.130 17.9311.00 72.97 C ATOM 1638 CG GLU A 1005 −24.759 50.844 16.561 1.00 75.62 CATOM 1639 CD GLU A 1005 −25.713 51.008 15.396 1.00 84.27 C ATOM 1640 OE1GLU A 1005 −26.925 50.899 15.640 1.00 91.81 O ATOM 1641 OE2 GLU A 1005−25.261 51.243 14.247 1.00 80.28 O ATOM 1642 N MET A 1006 −25.939 52.91320.453 1.00 71.72 N ATOM 1643 CA MET A 1006 −26.467 53.101 21.783 1.0072.95 C ATOM 1644 C MET A 1006 −27.562 54.182 21.796 1.00 73.35 C ATOM1645 O MET A 1006 −28.639 53.963 22.356 1.00 73.69 O ATOM 1646 CB MET A1006 −25.315 53.438 22.773 1.00 73.49 C ATOM 1647 CG MET A 1006 −25.76953.933 24.131 1.00 78.05 C ATOM 1648 SD MET A 1006 −24.447 54.468 25.2541.00 74.91 S ATOM 1649 CE MET A 1006 −24.293 53.083 26.396 1.00 86.03 CATOM 1650 N LEU A 1007 −27.286 55.335 21.179 1.00 72.95 N ATOM 1651 CALEU A 1007 −28.235 56.464 21.209 1.00 73.25 C ATOM 1652 C LEU A 1007−29.347 56.268 20.193 1.00 74.44 C ATOM 1653 O LEU A 1007 −30.441 56.79520.352 1.00 75.83 O ATOM 1654 CB LEU A 1007 −27.539 57.814 21.003 1.0071.83 C ATOM 1655 CG LEU A 1007 −26.953 58.593 22.197 1.00 76.11 C ATOM1656 CD1 LEU A 1007 −28.031 58.916 23.248 1.00 80.06 C ATOM 1657 CD2 LEUA 1007 −25.786 57.886 22.829 1.00 73.36 C ATOM 1658 N ARG A 1008 −29.06955.514 19.149 1.00 77.47 N ATOM 1659 CA ARG A 1008 −30.095 55.137 18.1831.00 77.36 C ATOM 1660 C ARG A 1008 −31.138 54.231 18.865 1.00 76.88 CATOM 1661 O ARG A 1008 −32.311 54.329 18.575 1.00 75.51 O ATOM 1662 CBARG A 1008 −29.437 54.430 17.040 1.00 72.89 C ATOM 1663 CG ARG A 1008−30.325 54.077 15.936 1.00 83.81 C ATOM 1664 CD ARG A 1008 −29.53053.460 14.840 1.00 80.34 C ATOM 1665 NE ARG A 1008 −30.368 52.534 14.0931.00 81.78 N ATOM 1666 CZ ARG A 1008 −29.944 51.431 13.485 1.00 85.98 CATOM 1667 NH1 ARG A 1008 −28.660 51.081 13.524 1.00 80.09 N ATOM 1668NH2 ARG A 1008 −30.823 50.660 12.823 1.00 93.61 N ATOM 1669 N ILE A 1009−30.678 53.355 19.773 1.00 78.10 N ATOM 1670 CA ILE A 1009 −31.54852.465 20.543 1.00 75.58 C ATOM 1671 C ILE A 1009 −32.251 53.251 21.6581.00 78.69 C ATOM 1672 O ILE A 1009 −33.455 53.134 21.826 1.00 81.42 OATOM 1673 CB ILE A 1009 −30.736 51.274 21.155 1.00 78.60 C ATOM 1674 CG1ILE A 1009 −30.220 50.329 20.057 1.00 70.72 C ATOM 1675 CG2 ILE A 1009−31.568 50.493 22.202 1.00 73.84 C ATOM 1676 CD1 ILE A 1009 −28.92449.579 20.447 1.00 68.79 C ATOM 1677 N ASP A 1010 −31.492 54.049 22.4121.00 81.55 N ATOM 1678 CA ASP A 1010 −32.048 54.813 23.555 1.00 80.99 CATOM 1679 C ASP A 1010 −32.892 56.045 23.199 1.00 82.41 C ATOM 1680 OASP A 1010 −33.892 56.302 23.876 1.00 83.53 O ATOM 1681 CB ASP A 1010−30.918 55.254 24.486 1.00 79.05 C ATOM 1682 CG ASP A 1010 −30.27354.103 25.216 1.00 81.30 C ATOM 1683 OD1 ASP A 1010 −30.918 53.07625.354 1.00 79.79 O ATOM 1684 OD2 ASP A 1010 −29.110 54.217 25.660 1.0068.53 O ATOM 1685 N GLU A 1011 −32.499 56.790 22.163 1.00 81.15 N ATOM1686 CA GLU A 1011 −33.206 58.046 21.749 1.00 82.09 C ATOM 1687 C GLU A1011 −34.075 57.898 20.499 1.00 82.84 C ATOM 1688 O GLU A 1011 −35.12458.541 20.397 1.00 86.08 O ATOM 1689 CB GLU A 1011 −32.189 59.197 21.5481.00 85.07 C ATOM 1690 CG GLU A 1011 −32.748 60.535 20.987 1.00 86.91 CATOM 1691 CD GLU A 1011 −33.834 61.166 21.857 1.00 92.06 C ATOM 1692 OE1GLU A 1011 −33.791 60.980 23.098 1.00 92.05 O ATOM 1693 OE2 GLU A 1011−34.736 61.855 21.291 1.00 82.39 O ATOM 1694 N GLY A 1012 −33.648 57.06719.550 1.00 77.24 N ATOM 1695 CA GLY A 1012 −34.407 56.839 18.336 1.0075.19 C ATOM 1696 C GLY A 1012 −33.925 57.768 17.246 1.00 74.64 C ATOM1697 O GLY A 1012 −33.635 58.934 17.494 1.00 71.63 O ATOM 1698 N LEU A1013 −33.841 57.245 16.032 1.00 74.33 N ATOM 1699 CA LEU A 1013 −33.40258.024 14.892 1.00 73.75 C ATOM 1700 C LEU A 1013 −34.653 58.394 14.1031.00 73.66 C ATOM 1701 O LEU A 1013 −35.397 57.511 13.670 1.00 72.82 OATOM 1702 CB LEU A 1013 −32.419 57.214 14.060 1.00 73.46 C ATOM 1703 CGLEU A 1013 −31.520 58.014 13.118 1.00 77.16 C ATOM 1704 CD1 LEU A 1013−30.400 57.161 12.611 1.00 81.09 C ATOM 1705 CD2 LEU A 1013 −32.30458.573 11.978 1.00 75.18 C ATOM 1706 N ARG A 1014 −34.875 59.696 13.9251.00 73.47 N ATOM 1707 CA ARG A 1014 −36.005 60.206 13.143 1.00 75.08 CATOM 1708 C ARG A 1014 −35.492 60.915 11.894 1.00 73.46 C ATOM 1709 OARG A 1014 −34.665 61.818 11.967 1.00 72.29 O ATOM 1710 CB ARG A 1014−36.899 61.098 13.994 1.00 73.94 C ATOM 1711 CG ARG A 1014 −37.55260.286 15.128 1.00 82.18 C ATOM 1712 CD ARG A 1014 −38.783 60.917 15.7161.00 87.29 C ATOM 1713 NE ARG A 1014 −39.879 61.089 14.746 1.00 93.86 NATOM 1714 CZ ARG A 1014 −41.129 61.469 15.053 1.00 92.93 C ATOM 1715 NH1ARG A 1014 −41.488 61.729 16.323 1.00 95.92 N ATOM 1716 NH2 ARG A 1014−42.038 61.591 14.080 1.00 92.74 N ATOM 1717 N LEU A 1015 −36.001 60.48110.746 1.00 73.79 N ATOM 1718 CA LEU A 1015 −35.566 60.969 9.444 1.0073.57 C ATOM 1719 C LEU A 1015 −36.284 62.239 9.033 1.00 74.92 C ATOM1720 O LEU A 1015 −35.757 62.988 8.224 1.00 72.14 O ATOM 1721 CB LEU A1015 −35.781 59.871 8.411 1.00 73.34 C ATOM 1722 CG LEU A 1015 −35.06558.555 8.749 1.00 71.48 C ATOM 1723 CD1 LEU A 1015 −35.240 57.576 7.6261.00 66.34 C ATOM 1724 CD2 LEU A 1015 −33.595 58.783 9.033 1.00 63.66 CATOM 1725 N LYS A 1016 −37.480 62.472 9.593 1.00 77.95 N ATOM 1726 CALYS A 1016 −38.274 63.678 9.327 1.00 75.82 C ATOM 1727 C LYS A 1016−38.338 64.571 10.563 1.00 73.96 C ATOM 1728 O LYS A 1016 −38.379 64.06511.680 1.00 71.82 O ATOM 1729 CB LYS A 1016 −39.688 63.302 8.908 1.0075.21 C ATOM 1730 CG LYS A 1016 −39.744 62.451 7.649 1.00 79.49 C ATOM1731 CD LYS A 1016 −41.159 62.214 7.176 1.00 77.17 C ATOM 1732 CE LYS A1016 −41.160 61.358 5.900 1.00 81.57 C ATOM 1733 NZ LYS A 1016 −42.52161.187 5.296 1.00 74.99 N ATOM 1734 N ILE A 1017 −38.347 65.889 10.3341.00 73.18 N ATOM 1735 CA ILE A 1017 −38.511 66.900 11.372 1.00 72.14 CATOM 1736 C ILE A 1017 −39.703 66.595 12.265 1.00 73.25 C ATOM 1737 OILE A 1017 −40.764 66.207 11.774 1.00 74.28 O ATOM 1738 CB ILE A 1017−38.705 68.320 10.765 1.00 71.53 C ATOM 1739 CG1 ILE A 1017 −37.39168.862 10.210 1.00 74.05 C ATOM 1740 CG2 ILE A 1017 −39.204 69.30811.788 1.00 70.34 C ATOM 1741 CD1 ILE A 1017 −37.443 70.318 9.833 1.0073.51 C ATOM 1742 N TYR A 1018 −39.515 66.773 13.573 1.00 72.91 N ATOM1743 CA TYR A 1018 −40.571 66.562 14.552 1.00 74.12 C ATOM 1744 C TYR A1018 −40.391 67.540 15.700 1.00 73.99 C ATOM 1745 O TYR A 1018 −39.30468.091 15.883 1.00 75.95 O ATOM 1746 CB TYR A 1018 −40.546 65.119 15.0701.00 75.31 C ATOM 1747 CG TYR A 1018 −39.316 64.769 15.884 1.00 75.01 CATOM 1748 CD1 TYR A 1018 −39.355 64.765 17.283 1.00 77.73 C ATOM 1749CD2 TYR A 1018 −38.113 64.440 15.263 1.00 75.41 C ATOM 1750 CE1 TYR A1018 −38.223 64.442 18.042 1.00 72.04 C ATOM 1751 CE2 TYR A 1018 −36.97464.116 16.016 1.00 77.21 C ATOM 1752 CZ TYR A 1018 −37.042 64.120 17.4031.00 76.64 C ATOM 1753 OH TYR A 1018 −35.923 63.800 18.142 1.00 79.46 OATOM 1754 N LYS A 1019 −41.461 67.746 16.459 1.00 73.71 N ATOM 1755 CALYS A 1019 −41.427 68.597 17.636 1.00 74.41 C ATOM 1756 C LYS A 1019−41.056 67.729 18.826 1.00 75.37 C ATOM 1757 O LYS A 1019 −41.715 66.71819.073 1.00 77.13 O ATOM 1758 CB LYS A 1019 −42.773 69.262 17.885 1.0073.28 C ATOM 1759 CG LYS A 1019 −43.104 70.352 16.899 1.00 72.01 C ATOM1760 CD LYS A 1019 −44.357 71.099 17.326 1.00 72.42 C ATOM 1761 CE LYS A1019 −44.649 72.280 16.414 1.00 73.72 C ATOM 1762 NZ LYS A 1019 −45.91072.993 16.796 1.00 71.74 N ATOM 1763 N ASP A 1020 −40.012 68.112 19.5611.00 76.71 N ATOM 1764 CA ASP A 1020 −39.610 67.388 20.776 1.00 78.54 CATOM 1765 C ASP A 1020 −40.629 67.615 21.913 1.00 78.53 C ATOM 1766 OASP A 1020 −41.693 68.205 21.698 1.00 79.88 O ATOM 1767 CB ASP A 1020−38.174 67.778 21.198 1.00 79.44 C ATOM 1768 CG ASP A 1020 −38.07169.183 21.838 1.00 81.53 C ATOM 1769 OD1 ASP A 1020 −39.087 69.87322.069 1.00 77.18 O ATOM 1770 OD2 ASP A 1020 −36.929 69.600 22.116 1.0089.87 O ATOM 1771 N THR A 1021 −40.297 67.147 23.112 1.00 81.31 N ATOM1772 CA THR A 1021 −41.163 67.303 24.299 1.00 81.40 C ATOM 1773 C THR A1021 −41.464 68.771 24.674 1.00 81.89 C ATOM 1774 O THR A 1021 −42.56269.085 25.152 1.00 83.48 O ATOM 1775 CB THR A 1021 −40.532 66.624 25.5141.00 83.23 C ATOM 1776 OG1 THR A 1021 −39.212 67.149 25.716 1.00 86.46 OATOM 1777 CG2 THR A 1021 −40.454 65.100 25.305 1.00 83.63 C ATOM 1778 NGLU A 1022 −40.488 69.653 24.447 1.00 81.25 N ATOM 1779 CA GLU A 1022−40.617 71.091 24.720 1.00 80.42 C ATOM 1780 C GLU A 1022 −41.256 71.86323.534 1.00 79.28 C ATOM 1781 O GLU A 1022 −41.339 73.093 23.576 1.0079.62 O ATOM 1782 CB GLU A 1022 −39.243 71.710 25.032 1.00 81.04 C ATOM1783 CG GLU A 1022 −38.301 70.940 26.011 1.00 82.43 C ATOM 1784 CD GLU A1022 −38.788 70.891 27.448 1.00 92.01 C ATOM 1785 OE1 GLU A 1022 −39.74971.611 27.806 1.00 94.63 O ATOM 1786 OE2 GLU A 1022 −38.192 70.11728.231 1.00 97.00 O ATOM 1787 N GLY A 1023 −41.696 71.146 22.491 1.0079.51 N ATOM 1788 CA GLY A 1023 −42.310 71.743 21.302 1.00 78.42 C ATOM1789 C GLY A 1023 −41.324 72.232 20.244 1.00 78.89 C ATOM 1790 O GLY A1023 −41.750 72.842 19.255 1.00 79.69 O ATOM 1791 N TYR A 1024 −40.02171.971 20.436 1.00 77.49 N ATOM 1792 CA TYR A 1024 −38.981 72.476 19.5321.00 76.94 C ATOM 1793 C TYR A 1024 −38.694 71.548 18.363 1.00 77.71 CATOM 1794 O TYR A 1024 −38.686 70.321 18.524 1.00 79.61 O ATOM 1795 CBTYR A 1024 −37.663 72.699 20.269 1.00 77.27 C ATOM 1796 CG TYR A 1024−37.711 73.692 21.400 1.00 79.68 C ATOM 1797 CD1 TYR A 1024 −38.14574.987 21.192 1.00 81.27 C ATOM 1798 CD2 TYR A 1024 −37.313 73.33722.684 1.00 79.56 C ATOM 1799 CE1 TYR A 1024 −38.189 75.913 22.232 1.0083.68 C ATOM 1800 CE2 TYR A 1024 −37.353 74.253 23.736 1.00 78.83 C ATOM1801 CZ TYR A 1024 −37.792 75.541 23.503 1.00 80.59 C ATOM 1802 OH TYR A1024 −37.836 76.457 24.534 1.00 83.58 O ATOM 1803 N TYR A 1025 −38.45272.145 17.191 1.00 74.53 N ATOM 1804 CA TYR A 1025 −38.176 71.387 15.9691.00 73.53 C ATOM 1805 C TYR A 1025 −36.849 70.657 16.050 1.00 68.58 CATOM 1806 O TYR A 1025 −35.801 71.285 16.188 1.00 69.06 O ATOM 1807 CBTYR A 1025 −38.195 72.295 14.736 1.00 71.74 C ATOM 1808 CG TYR A 1025−39.564 72.806 14.416 1.00 70.34 C ATOM 1809 CD1 TYR A 1025 −39.92474.104 14.717 1.00 71.87 C ATOM 1810 CD2 TYR A 1025 −40.510 71.98313.810 1.00 71.28 C ATOM 1811 CE1 TYR A 1025 −41.186 74.577 14.423 1.0077.57 C ATOM 1812 CE2 TYR A 1025 −41.783 72.445 13.509 1.00 70.46 C ATOM1813 CZ TYR A 1025 −42.118 73.747 13.819 1.00 72.50 C ATOM 1814 OH TYR A1025 −43.366 74.222 13.531 1.00 73.02 O ATOM 1815 N THR A 1026 −36.92369.330 15.960 1.00 68.83 N ATOM 1816 CA THR A 1026 −35.783 68.431 16.1291.00 69.83 C ATOM 1817 C THR A 1026 −35.730 67.408 15.000 1.00 71.01 CATOM 1818 O THR A 1026 −36.752 67.153 14.338 1.00 75.69 O ATOM 1819 CBTHR A 1026 −35.922 67.741 17.500 1.00 68.73 C ATOM 1820 OG1 THR A 1026−36.033 68.752 18.502 1.00 71.36 O ATOM 1821 CG2 THR A 1026 −34.75066.824 17.843 1.00 65.86 C ATOM 1822 N ILE A 1027 −34.552 66.825 14.7691.00 69.80 N ATOM 1823 CA ILE A 1027 −34.397 65.788 13.737 1.00 72.31 CATOM 1824 C ILE A 1027 −33.259 64.836 14.091 1.00 73.35 C ATOM 1825 OILE A 1027 −32.339 65.215 14.794 1.00 74.44 O ATOM 1826 CB ILE A 1027−34.153 66.428 12.350 1.00 71.44 C ATOM 1827 CG1 ILE A 1027 −34.48265.443 11.224 1.00 73.60 C ATOM 1828 CG2 ILE A 1027 −32.713 66.93412.235 1.00 69.68 C ATOM 1829 CD1 ILE A 1027 −34.540 66.100 9.855 1.0069.11 C ATOM 1830 N GLY A 1028 −33.331 63.596 13.601 1.00 72.50 N ATOM1831 CA GLY A 1028 −32.287 62.599 13.863 1.00 71.73 C ATOM 1832 C GLY A1028 −32.366 62.107 15.292 1.00 71.31 C ATOM 1833 O GLY A 1028 −33.45561.891 15.807 1.00 70.02 O ATOM 1834 N ILE A 1029 −31.202 61.935 15.9181.00 73.01 N ATOM 1835 CA ILE A 1029 −31.090 61.460 17.295 1.00 72.52 CATOM 1836 C ILE A 1029 −31.167 62.687 18.232 1.00 74.59 C ATOM 1837 OILE A 1029 −30.158 63.152 18.782 1.00 77.27 O ATOM 1838 CB ILE A 1029−29.786 60.637 17.526 1.00 72.24 C ATOM 1839 CG1 ILE A 1029 −29.69659.446 16.569 1.00 71.48 C ATOM 1840 CG2 ILE A 1029 −29.716 60.14018.998 1.00 70.08 C ATOM 1841 CD1 ILE A 1029 −28.290 58.898 16.365 1.0072.58 C ATOM 1842 N GLY A 1030 −32.378 63.195 18.397 1.00 72.49 N ATOM1843 CA GLY A 1030 −32.622 64.375 19.197 1.00 72.04 C ATOM 1844 C GLY A1030 −31.760 65.582 18.861 1.00 73.16 C ATOM 1845 O GLY A 1030 −31.34966.291 19.755 1.00 72.16 O ATOM 1846 N HIS A 1031 −31.480 65.821 17.5741.00 75.22 N ATOM 1847 CA HIS A 1031 −30.705 67.007 17.192 1.00 74.36 CATOM 1848 C HIS A 1031 −31.668 68.175 17.134 1.00 75.22 C ATOM 1849 OHIS A 1031 −32.614 68.137 16.371 1.00 78.88 O ATOM 1850 CB HIS A 1031−29.985 66.864 15.840 1.00 76.09 C ATOM 1851 CG HIS A 1031 −29.19068.068 15.470 1.00 74.73 C ATOM 1852 ND1 HIS A 1031 −27.828 68.14015.644 1.00 73.37 N ATOM 1853 CD2 HIS A 1031 −29.568 69.255 14.941 1.0070.52 C ATOM 1854 CE1 HIS A 1031 −27.397 69.320 15.234 1.00 72.05 C ATOM1855 NE2 HIS A 1031 −28.434 70.016 14.805 1.00 73.60 N ATOM 1856 N LEU A1032 −31.420 69.209 17.937 1.00 74.86 N ATOM 1857 CA LEU A 1032 −32.27170.400 17.966 1.00 75.57 C ATOM 1858 C LEU A 1032 −31.871 71.334 16.8401.00 75.37 C ATOM 1859 O LEU A 1032 −30.681 71.654 16.690 1.00 76.52 OATOM 1860 CB LEU A 1032 −32.148 71.137 19.305 1.00 74.42 C ATOM 1861 CGLEU A 1032 −33.092 72.326 19.545 1.00 76.35 C ATOM 1862 CD1 LEU A 1032−34.490 71.821 19.722 1.00 81.83 C ATOM 1863 CD2 LEU A 1032 −32.68273.124 20.760 1.00 73.47 C ATOM 1864 N LEU A 1033 −32.857 71.770 16.0571.00 74.07 N ATOM 1865 CA LEU A 1033 −32.610 72.675 14.928 1.00 73.31 CATOM 1866 C LEU A 1033 −32.684 74.147 15.331 1.00 72.89 C ATOM 1867 OLEU A 1033 −31.789 74.937 15.003 1.00 70.82 O ATOM 1868 CB LEU A 1033−33.595 72.374 13.787 1.00 73.71 C ATOM 1869 CG LEU A 1033 −33.38071.032 13.099 1.00 68.93 C ATOM 1870 CD1 LEU A 1033 −34.527 70.72712.146 1.00 71.95 C ATOM 1871 CD2 LEU A 1033 −32.023 71.025 12.372 1.0066.34 C ATOM 1872 N THR A 1034 −33.751 74.502 16.039 1.00 73.89 N ATOM1873 CA THR A 1034 −33.986 75.873 16.494 1.00 71.83 C ATOM 1874 C THR A1034 −35.070 75.849 17.538 1.00 73.47 C ATOM 1875 O THR A 1034 −35.84974.896 17.591 1.00 74.91 O ATOM 1876 CB THR A 1034 −34.439 76.792 15.3251.00 71.67 C ATOM 1877 OG1 THR A 1034 −34.731 78.108 15.807 1.00 67.14 OATOM 1878 CG2 THR A 1034 −35.685 76.231 14.634 1.00 67.88 C ATOM 1879 NLYS A 1035 −35.120 76.892 18.365 1.00 73.16 N ATOM 1880 CA LYS A 1035−36.190 77.037 19.352 1.00 74.56 C ATOM 1881 C LYS A 1035 −37.402 77.82418.786 1.00 74.05 C ATOM 1882 O LYS A 1035 −38.435 77.955 19.456 1.0073.29 O ATOM 1883 CB LYS A 1035 −35.669 77.666 20.656 1.00 75.36 C ATOM1884 CG LYS A 1035 −34.711 76.751 21.441 1.00 74.77 C ATOM 1885 CD LYS A1035 −34.455 77.236 22.866 1.00 77.28 C ATOM 1886 CE LYS A 1035 −33.83678.641 22.924 1.00 82.03 C ATOM 1887 NZ LYS A 1035 −33.484 79.060 24.3311.00 81.98 N ATOM 1888 N SER A 1036 −37.256 78.330 17.559 1.00 73.73 NATOM 1889 CA SER A 1036 −38.296 79.089 16.869 1.00 73.96 C ATOM 1890 CSER A 1036 −39.540 78.248 16.553 1.00 73.14 C ATOM 1891 O SER A 1036−39.420 77.055 16.331 1.00 75.32 O ATOM 1892 CB SER A 1036 −37.71379.649 15.561 1.00 74.91 C ATOM 1893 OG SER A 1036 −38.688 80.260 14.7431.00 72.52 O ATOM 1894 N PRO A 1037 −40.740 78.874 16.532 1.00 73.94 NATOM 1895 CA PRO A 1037 −41.968 78.169 16.153 1.00 73.36 C ATOM 1896 CPRO A 1037 −42.157 78.061 14.632 1.00 74.22 C ATOM 1897 O PRO A 1037−43.072 77.365 14.184 1.00 76.00 O ATOM 1898 CB PRO A 1037 −43.05479.058 16.732 1.00 72.94 C ATOM 1899 CG PRO A 1037 −42.497 80.415 16.5881.00 73.18 C ATOM 1900 CD PRO A 1037 −41.032 80.284 16.861 1.00 73.91 CATOM 1901 N SER A 1038 −41.308 78.744 13.855 1.00 73.88 N ATOM 1902 CASER A 1038 −41.346 78.688 12.406 1.00 73.57 C ATOM 1903 C SER A 1038−40.709 77.399 11.934 1.00 74.14 C ATOM 1904 O SER A 1038 −39.552 77.12212.271 1.00 73.16 O ATOM 1905 CB SER A 1038 −40.594 79.872 11.797 1.0073.20 C ATOM 1906 OG SER A 1038 −40.486 79.739 10.391 1.00 72.62 O ATOM1907 N LEU A 1039 −41.463 76.617 11.163 1.00 72.03 N ATOM 1908 CA LEU A1039 −40.961 75.378 10.590 1.00 72.88 C ATOM 1909 C LEU A 1039 −39.90575.704 9.557 1.00 72.75 C ATOM 1910 O LEU A 1039 −38.836 75.100 9.5431.00 74.58 O ATOM 1911 CB LEU A 1039 −42.092 74.581 9.944 1.00 72.16 CATOM 1912 CG LEU A 1039 −41.717 73.267 9.243 1.00 71.92 C ATOM 1913 CD1LEU A 1039 −41.053 72.299 10.198 1.00 68.87 C ATOM 1914 CD2 LEU A 1039−42.951 72.627 8.601 1.00 71.51 C ATOM 1915 N ASN A 1040 −40.218 76.6658.694 1.00 74.45 N ATOM 1916 CA ASN A 1040 −39.282 77.148 7.665 1.0074.48 C ATOM 1917 C ASN A 1040 −37.918 77.589 8.246 1.00 74.80 C ATOM1918 O ASN A 1040 −36.899 77.499 7.561 1.00 76.00 O ATOM 1919 CB ASN A1040 −39.937 78.277 6.838 1.00 74.13 C ATOM 1920 CG ASN A 1040 −41.13577.779 5.985 1.00 73.14 C ATOM 1921 OD1 ASN A 1040 −41.183 76.619 5.5551.00 70.14 O ATOM 1922 ND2 ASN A 1040 −42.095 78.665 5.748 1.00 66.32 NATOM 1923 N ALA A 1041 −37.918 78.058 9.500 1.00 74.86 N ATOM 1924 CAALA A 1041 −36.689 78.402 10.228 1.00 75.02 C ATOM 1925 C ALA A 1041−35.886 77.139 10.604 1.00 75.73 C ATOM 1926 O ALA A 1041 −34.664 77.15510.555 1.00 75.98 O ATOM 1927 CB ALA A 1041 −37.011 79.220 11.466 1.0073.60 C ATOM 1928 N ALA A 1042 −36.583 76.061 10.975 1.00 75.31 N ATOM1929 CA ALA A 1042 −35.953 74.762 11.236 1.00 74.13 C ATOM 1930 C ALA A1042 −35.329 74.212 9.956 1.00 75.51 C ATOM 1931 O ALA A 1042 −34.15273.844 9.946 1.00 75.22 O ATOM 1932 CB ALA A 1042 −36.969 73.782 11.7791.00 73.73 C ATOM 1933 N LYS A 1043 −36.135 74.164 8.887 1.00 75.74 NATOM 1934 CA LYS A 1043 −35.689 73.728 7.547 1.00 76.54 C ATOM 1935 CLYS A 1043 −34.445 74.481 7.067 1.00 76.74 C ATOM 1936 O LYS A 1043−33.526 73.869 6.530 1.00 78.09 O ATOM 1937 CB LYS A 1043 −36.823 73.8936.527 1.00 75.43 C ATOM 1938 CG LYS A 1043 −37.946 72.879 6.697 1.0076.23 C ATOM 1939 CD LYS A 1043 −39.136 73.199 5.806 1.00 82.58 C ATOM1940 CE LYS A 1043 −40.094 72.010 5.671 1.00 85.26 C ATOM 1941 NZ LYS A1043 −40.465 71.420 6.980 1.00 90.37 N ATOM 1942 N SER A 1044 −34.43075.798 7.267 1.00 75.27 N ATOM 1943 CA SER A 1044 −33.272 76.626 6.9311.00 75.10 C ATOM 1944 C SER A 1044 −32.023 76.175 7.694 1.00 75.62 CATOM 1945 O SER A 1044 −30.957 76.023 7.097 1.00 77.80 O ATOM 1946 CBSER A 1044 −33.557 78.096 7.232 1.00 75.53 C ATOM 1947 OG SER A 1044−32.465 78.910 6.848 1.00 75.67 O ATOM 1948 N GLU A 1045 −32.166 75.9669.006 1.00 73.82 N ATOM 1949 CA GLU A 1045 −31.062 75.471 9.836 1.0073.20 C ATOM 1950 C GLU A 1045 −30.641 74.064 9.456 1.00 71.02 C ATOM1951 O GLU A 1045 −29.448 73.752 9.494 1.00 71.07 O ATOM 1952 CB GLU A1045 −31.427 75.494 11.331 1.00 71.62 C ATOM 1953 CG GLU A 1045 −31.65976.884 11.923 1.00 73.11 C ATOM 1954 CD GLU A 1045 −30.431 77.762 11.9491.00 76.79 C ATOM 1955 OE1 GLU A 1045 −29.308 77.260 11.747 1.00 80.04 OATOM 1956 OE2 GLU A 1045 −30.595 78.980 12.177 1.00 85.26 O ATOM 1957 NLEU A 1046 −31.615 73.224 9.100 1.00 70.49 N ATOM 1958 CA LEU A 1046−31.342 71.839 8.710 1.00 73.29 C ATOM 1959 C LEU A 1046 −30.492 71.8077.455 1.00 73.52 C ATOM 1960 O LEU A 1046 −29.453 71.144 7.434 1.0074.73 O ATOM 1961 CB LEU A 1046 −32.644 71.064 8.469 1.00 75.19 C ATOM1962 CG LEU A 1046 −32.556 69.562 8.130 1.00 73.72 C ATOM 1963 CD1 LEU A1046 −31.881 68.775 9.243 1.00 74.36 C ATOM 1964 CD2 LEU A 1046 −33.94269.000 7.851 1.00 73.43 C ATOM 1965 N ASP A 1047 −30.945 72.529 6.4261.00 72.94 N ATOM 1966 CA ASP A 1047 −30.225 72.645 5.148 1.00 75.39 CATOM 1967 C ASP A 1047 −28.812 73.161 5.343 1.00 74.85 C ATOM 1968 O ASPA 1047 −27.875 72.634 4.758 1.00 76.38 O ATOM 1969 CB ASP A 1047 −30.95373.595 4.160 1.00 74.34 C ATOM 1970 CG ASP A 1047 −32.327 73.076 3.7131.00 75.72 C ATOM 1971 OD1 ASP A 1047 −32.707 71.938 4.072 1.00 72.63 OATOM 1972 OD2 ASP A 1047 −33.029 73.824 2.995 1.00 80.04 O ATOM 1973 NLYS A 1048 −28.677 74.193 6.168 1.00 75.86 N ATOM 1974 CA LYS A 1048−27.379 74.779 6.494 1.00 75.59 C ATOM 1975 C LYS A 1048 −26.464 73.7797.248 1.00 75.44 C ATOM 1976 O LYS A 1048 −25.241 73.810 7.088 1.0075.09 O ATOM 1977 CB LYS A 1048 −27.603 76.061 7.296 1.00 75.61 C ATOM1978 CG LYS A 1048 −26.368 76.896 7.555 1.00 77.43 C ATOM 1979 CD LYS A1048 −26.729 78.377 7.838 1.00 78.34 C ATOM 1980 CE LYS A 1048 −27.67178.567 9.048 1.00 80.49 C ATOM 1981 NZ LYS A 1048 −27.074 78.073 10.3221.00 79.34 N ATOM 1982 N ALA A 1049 −27.073 72.903 8.055 1.00 73.58 NATOM 1983 CA ALA A 1049 −26.345 71.870 8.804 1.00 74.82 C ATOM 1984 CALA A 1049 −25.950 70.699 7.909 1.00 75.13 C ATOM 1985 O ALA A 1049−24.797 70.271 7.913 1.00 75.27 O ATOM 1986 CB ALA A 1049 −27.188 71.3649.973 1.00 73.33 C ATOM 1987 N ILE A 1050 −26.924 70.194 7.152 1.0073.31 N ATOM 1988 CA ILE A 1050 −26.746 69.038 6.255 1.00 73.24 C ATOM1989 C ILE A 1050 −25.994 69.378 4.951 1.00 72.49 C ATOM 1990 O ILE A1050 −25.155 68.602 4.500 1.00 72.34 O ATOM 1991 CB ILE A 1050 −28.12468.397 5.931 1.00 73.95 C ATOM 1992 CG1 ILE A 1050 −28.758 67.828 7.2071.00 72.34 C ATOM 1993 CG2 ILE A 1050 −27.996 67.297 4.907 1.00 70.31 CATOM 1994 CD1 ILE A 1050 −27.968 66.650 7.819 1.00 75.79 C ATOM 1995 NGLY A 1051 −26.304 70.530 4.362 1.00 70.45 N ATOM 1996 CA GLY A 1051−25.628 71.022 3.152 1.00 69.29 C ATOM 1997 C GLY A 1051 −26.371 70.8101.841 1.00 68.84 C ATOM 1998 O GLY A 1051 −25.741 70.624 0.800 1.0064.99 O ATOM 1999 N ARG A 1052 −27.703 70.841 1.900 1.00 66.56 N ATOM2000 CA ARG A 1052 −28.563 70.680 0.730 1.00 67.90 C ATOM 2001 C ARG A1052 −29.995 71.012 1.090 1.00 68.30 C ATOM 2002 O ARG A 1052 −30.30971.175 2.261 1.00 71.80 O ATOM 2003 CB ARG A 1052 −28.502 69.251 0.1621.00 66.98 C ATOM 2004 CG ARG A 1052 −28.971 68.148 1.076 1.00 72.44 CATOM 2005 CD ARG A 1052 −29.034 66.833 0.330 1.00 70.28 C ATOM 2006 NEARG A 1052 −29.469 65.745 1.210 1.00 74.46 N ATOM 2007 CZ ARG A 1052−30.740 65.449 1.540 1.00 75.81 C ATOM 2008 NH1 ARG A 1052 −31.77266.146 1.076 1.00 75.31 N ATOM 2009 NH2 ARG A 1052 −30.991 64.430 2.3541.00 78.34 N ATOM 2010 N ASN A 1053 −30.856 71.108 0.083 1.00 67.63 NATOM 2011 CA ASN A 1053 −32.289 71.307 0.299 1.00 69.73 C ATOM 2012 CASN A 1053 −32.876 69.984 0.719 1.00 69.35 C ATOM 2013 O ASN A 1053−33.005 69.075 −0.095 1.00 69.80 O ATOM 2014 CB ASN A 1053 −32.99671.829 −0.957 1.00 70.79 C ATOM 2015 CG ASN A 1053 −32.759 73.310 −1.2011.00 72.54 C ATOM 2016 OD1 ASN A 1053 −33.223 73.847 −2.196 1.00 78.00 OATOM 2017 ND2 ASN A 1053 −32.037 73.975 −0.292 1.00 81.34 N ATOM 2018 NTHR A 1054 −33.228 69.880 1.996 1.00 72.38 N ATOM 2019 CA THR A 1054−33.753 68.647 2.552 1.00 72.53 C ATOM 2020 C THR A 1054 −35.266 68.5882.510 1.00 73.02 C ATOM 2021 O THR A 1054 −35.826 67.516 2.305 1.0077.21 O ATOM 2022 CB THR A 1054 −33.251 68.458 3.982 1.00 71.32 C ATOM2023 OG1 THR A 1054 −33.557 69.619 4.758 1.00 71.59 O ATOM 2024 CG2 THRA 1054 −31.748 68.254 3.981 1.00 73.10 C ATOM 2025 N ASN A 1055 −35.90869.737 2.702 1.00 75.24 N ATOM 2026 CA ASN A 1055 −37.360 69.842 2.8151.00 76.78 C ATOM 2027 C ASN A 1055 −37.876 69.053 4.046 1.00 76.94 CATOM 2028 O ASN A 1055 −38.985 68.522 4.044 1.00 82.59 O ATOM 2029 CBASN A 1055 −38.062 69.398 1.517 1.00 76.21 C ATOM 2030 CG ASN A 1055−39.511 69.867 1.431 1.00 78.54 C ATOM 2031 OD1 ASN A 1055 −39.93070.812 2.109 1.00 77.71 O ATOM 2032 ND2 ASN A 1055 −40.285 69.199 0.5871.00 87.37 N ATOM 2033 N GLY A 1056 −37.054 68.984 5.091 1.00 76.49 NATOM 2034 CA GLY A 1056 −37.403 68.283 6.314 1.00 76.60 C ATOM 2035 CGLY A 1056 −36.995 66.832 6.409 1.00 74.80 C ATOM 2036 O GLY A 1056−37.028 66.298 7.489 1.00 77.03 O ATOM 2037 N VAL A 1057 −36.610 66.1925.301 1.00 74.21 N ATOM 2038 CA VAL A 1057 −36.246 64.770 5.320 1.0073.68 C ATOM 2039 C VAL A 1057 −34.745 64.554 5.095 1.00 73.15 C ATOM2040 O VAL A 1057 −34.181 65.114 4.151 1.00 77.13 O ATOM 2041 CB VAL A1057 −37.029 63.981 4.261 1.00 72.56 C ATOM 2042 CG1 VAL A 1057 −36.71162.498 4.368 1.00 74.94 C ATOM 2043 CG2 VAL A 1057 −38.523 64.201 4.4321.00 70.86 C ATOM 2044 N ILE A 1058 −34.120 63.742 5.963 1.00 71.83 NATOM 2045 CA ILE A 1058 −32.698 63.347 5.843 1.00 71.92 C ATOM 2046 CILE A 1058 −32.520 61.833 5.872 1.00 73.30 C ATOM 2047 O ILE A 1058−33.382 61.111 6.370 1.00 76.64 O ATOM 2048 CB ILE A 1058 −31.815 63.9536.956 1.00 67.96 C ATOM 2049 CG1 ILE A 1058 −32.220 63.448 8.364 1.0071.45 C ATOM 2050 CG2 ILE A 1058 −31.883 65.457 6.899 1.00 65.81 C ATOM2051 CD1 ILE A 1058 −31.205 63.816 9.445 1.00 65.73 C ATOM 2052 N THR A1059 −31.392 61.362 5.343 1.00 72.25 N ATOM 2053 CA THR A 1059 −31.07059.932 5.345 1.00 71.10 C ATOM 2054 C THR A 1059 −30.533 59.545 6.7121.00 70.11 C ATOM 2055 O THR A 1059 −30.166 60.405 7.495 1.00 71.12 OATOM 2056 CB THR A 1059 −29.990 59.595 4.353 1.00 70.42 C ATOM 2057 OG1THR A 1059 −28.775 60.228 4.776 1.00 67.63 O ATOM 2058 CG2 THR A 1059−30.377 60.056 2.874 1.00 63.19 C ATOM 2059 N LYS A 1060 −30.493 58.2446.972 1.00 70.88 N ATOM 2060 CA LYS A 1060 −29.950 57.684 8.208 1.0071.95 C ATOM 2061 C LYS A 1060 −28.481 58.087 8.416 1.00 72.94 C ATOM2062 O LYS A 1060 −28.082 58.452 9.536 1.00 71.54 O ATOM 2063 CB LYS A1060 −30.074 56.151 8.215 1.00 70.63 C ATOM 2064 CG LYS A 1060 −29.32555.466 9.387 1.00 75.49 C ATOM 2065 CD LYS A 1060 −29.500 53.953 9.4191.00 73.88 C ATOM 2066 CE LYS A 1060 −28.422 53.322 10.308 1.00 71.79 CATOM 2067 NZ LYS A 1060 −28.584 51.848 10.427 1.00 80.16 N ATOM 2068 NASP A 1061 −27.706 58.010 7.330 1.00 68.84 N ATOM 2069 CA ASP A 1061−26.282 58.339 7.320 1.00 68.20 C ATOM 2070 C ASP A 1061 −26.043 59.8147.586 1.00 66.58 C ATOM 2071 O ASP A 1061 −25.076 60.148 8.255 1.0070.66 O ATOM 2072 CB ASP A 1061 −25.622 57.923 5.983 1.00 64.64 C ATOM2073 CG ASP A 1061 −25.572 56.386 5.788 1.00 73.92 C ATOM 2074 OD1 ASP A1061 −25.501 55.619 6.782 1.00 83.01 O ATOM 2075 OD2 ASP A 1061 −25.60355.935 4.630 1.00 82.92 O ATOM 2076 N GLU A 1062 −26.909 60.687 7.0711.00 60.10 N ATOM 2077 CA GLU A 1062 −26.856 62.113 7.437 1.00 62.56 CATOM 2078 C GLU A 1062 −27.203 62.317 8.955 1.00 64.45 C ATOM 2079 O GLUA 1062 −26.559 63.127 9.619 1.00 62.28 O ATOM 2080 CB GLU A 1062 −27.77062.910 6.572 1.00 60.74 C ATOM 2081 CG GLU A 1062 −27.243 63.018 5.1421.00 60.93 C ATOM 2082 CD GLU A 1062 −28.230 63.606 4.198 1.00 68.90 CATOM 2083 OE1 GLU A 1062 −29.446 63.498 4.488 1.00 67.42 O ATOM 2084 OE2GLU A 1062 −27.795 64.182 3.155 1.00 73.08 O ATOM 2085 N ALA A 1063−28.197 61.590 9.485 1.00 64.40 N ATOM 2086 CA ALA A 1063 −28.494 61.64710.955 1.00 68.01 C ATOM 2087 C ALA A 1063 −27.251 61.296 11.748 1.0069.72 C ATOM 2088 O ALA A 1063 −26.948 61.940 12.745 1.00 79.20 O ATOM2089 CB ALA A 1063 −29.637 60.723 11.331 1.00 65.57 C ATOM 2090 N GLU A1064 −26.529 60.269 11.298 1.00 73.91 N ATOM 2091 CA GLU A 1064 −25.26159.873 11.920 1.00 71.50 C ATOM 2092 C GLU A 1064 −24.218 60.973 11.8151.00 72.04 C ATOM 2093 O GLU A 1064 −23.465 61.158 12.747 1.00 76.73 OATOM 2094 CB GLU A 1064 −24.746 58.584 11.294 1.00 70.30 C ATOM 2095 CGGLU A 1064 −23.391 58.090 11.772 1.00 71.10 C ATOM 2096 CD GLU A 1064−23.012 56.715 11.195 1.00 81.17 C ATOM 2097 OE1 GLU A 1064 −23.71156.194 10.251 1.00 87.16 O ATOM 2098 OE2 GLU A 1064 −22.006 56.15011.688 1.00 89.77 O ATOM 2099 N LYS A 1065 −24.168 61.701 10.695 1.0073.67 N ATOM 2100 CA LYS A 1065 −23.204 62.821 10.568 1.00 73.69 C ATOM2101 C LYS A 1065 −23.466 63.903 11.620 1.00 70.10 C ATOM 2102 O LYS A1065 −22.553 64.325 12.310 1.00 68.11 O ATOM 2103 CB LYS A 1065 −23.23263.461 9.190 1.00 75.31 C ATOM 2104 CG LYS A 1065 −22.891 62.546 8.0221.00 80.59 C ATOM 2105 CD LYS A 1065 −21.422 62.223 7.938 1.00 85.80 CATOM 2106 CE LYS A 1065 −21.098 61.379 6.680 1.00 81.37 C ATOM 2107 NZLYS A 1065 −21.661 61.944 5.425 1.00 72.55 N ATOM 2108 N LEU A 1066−24.718 64.335 11.723 1.00 69.54 N ATOM 2109 CA LEU A 1066 −25.13365.305 12.748 1.00 71.70 C ATOM 2110 C LEU A 1066 −24.854 64.804 14.1651.00 70.08 C ATOM 2111 O LEU A 1066 −24.476 65.572 15.033 1.00 68.08 OATOM 2112 CB LEU A 1066 −26.621 65.605 12.636 1.00 71.78 C ATOM 2113 CGLEU A 1066 −27.056 66.466 11.468 1.00 76.18 C ATOM 2114 CD1 LEU A 1066−28.565 66.357 11.261 1.00 78.42 C ATOM 2115 CD2 LEU A 1066 −26.62567.899 11.715 1.00 77.95 C ATOM 2116 N PHE A 1067 −25.049 63.513 14.3731.00 71.17 N ATOM 2117 CA PHE A 1067 −24.844 62.899 15.673 1.00 71.75 CATOM 2118 C PHE A 1067 −23.383 62.902 16.104 1.00 71.56 C ATOM 2119 OPHE A 1067 −23.098 63.171 17.275 1.00 76.85 O ATOM 2120 CB PHE A 1067−25.378 61.480 15.669 1.00 74.38 C ATOM 2121 CG PHE A 1067 −25.04660.740 16.880 1.00 75.45 C ATOM 2122 CD1 PHE A 1067 −25.810 60.89718.012 1.00 78.29 C ATOM 2123 CD2 PHE A 1067 −23.957 59.877 16.909 1.0081.22 C ATOM 2124 CE1 PHE A 1067 −25.509 60.221 19.138 1.00 77.84 C ATOM2125 CE2 PHE A 1067 −23.650 59.194 18.043 1.00 81.99 C ATOM 2126 CZ PHEA 1067 −24.425 59.361 19.165 1.00 78.68 C ATOM 2127 N ASN A 1068 −22.46462.611 15.188 1.00 72.06 N ATOM 2128 CA ASN A 1068 −21.035 62.658 15.5201.00 72.87 C ATOM 2129 C ASN A 1068 −20.611 64.092 15.888 1.00 74.75 CATOM 2130 O ASN A 1068 −19.722 64.272 16.716 1.00 76.41 O ATOM 2131 CBASN A 1068 −20.134 62.151 14.400 1.00 70.30 C ATOM 2132 CG ASN A 1068−20.477 60.741 13.936 1.00 77.74 C ATOM 2133 OD1 ASN A 1068 −20.90259.903 14.722 1.00 71.67 O ATOM 2134 ND2 ASN A 1068 −20.286 60.47912.617 1.00 72.67 N ATOM 2135 N GLN A 1069 −21.244 65.094 15.275 1.0073.62 N ATOM 2136 CA GLN A 1069 −20.982 66.487 15.631 1.00 73.62 C ATOM2137 C GLN A 1069 −21.541 66.803 17.020 1.00 73.06 C ATOM 2138 O GLN A1069 −20.874 67.455 17.802 1.00 68.92 O ATOM 2139 CB GLN A 1069 −21.58667.449 14.634 1.00 71.42 C ATOM 2140 CG GLN A 1069 −20.967 67.379 13.2491.00 81.86 C ATOM 2141 CD GLN A 1069 −21.717 68.235 12.239 1.00 81.55 CATOM 2142 OE1 GLN A 1069 −22.504 69.116 12.614 1.00 87.86 O ATOM 2143NE2 GLN A 1069 −21.480 67.980 10.948 1.00 91.37 N ATOM 2144 N ASP A 1070−22.759 66.341 17.304 1.00 69.64 N ATOM 2145 CA ASP A 1070 −23.36266.549 18.615 1.00 70.15 C ATOM 2146 C ASP A 1070 −22.510 65.879 19.6891.00 70.56 C ATOM 2147 O ASP A 1070 −22.272 66.486 20.737 1.00 71.26 OATOM 2148 CB ASP A 1070 −24.795 66.038 18.664 1.00 68.69 C ATOM 2149 CGASP A 1070 −25.742 66.828 17.756 1.00 77.13 C ATOM 2150 OD1 ASP A 1070−25.444 67.997 17.392 1.00 75.90 O ATOM 2151 OD2 ASP A 1070 −26.79466.277 17.400 1.00 74.40 O ATOM 2152 N VAL A 1071 −22.058 64.649 19.4261.00 67.27 N ATOM 2153 CA VAL A 1071 −21.150 63.948 20.341 1.00 69.23 CATOM 2154 C VAL A 1071 −19.906 64.780 20.577 1.00 70.51 C ATOM 2155 OVAL A 1071 −19.509 64.968 21.714 1.00 71.56 O ATOM 2156 CB VAL A 1071−20.737 62.563 19.820 1.00 66.16 C ATOM 2157 CG1 VAL A 1071 −19.56162.086 20.548 1.00 63.68 C ATOM 2158 CG2 VAL A 1071 −21.887 61.57019.952 1.00 68.51 C ATOM 2159 N ASP A 1072 −19.294 65.280 19.500 1.0074.44 N ATOM 2160 CA ASP A 1072 −18.128 66.154 19.623 1.00 73.07 C ATOM2161 C ASP A 1072 −18.410 67.321 20.555 1.00 74.85 C ATOM 2162 O ASP A1072 −17.628 67.585 21.464 1.00 78.47 O ATOM 2163 CB ASP A 1072 −17.68866.703 18.269 1.00 77.99 C ATOM 2164 CG ASP A 1072 −16.502 67.645 18.3871.00 82.49 C ATOM 2165 OD1 ASP A 1072 −15.355 67.156 18.521 1.00 93.79 OATOM 2166 OD2 ASP A 1072 −16.730 68.874 18.341 1.00 88.07 O ATOM 2167 NALA A 1073 −19.530 68.003 20.316 1.00 70.34 N ATOM 2168 CA ALA A 1073−19.981 69.126 21.153 1.00 71.13 C ATOM 2169 C ALA A 1073 −20.178 68.74222.643 1.00 72.31 C ATOM 2170 O ALA A 1073 −19.771 69.495 23.520 1.0072.64 O ATOM 2171 CB ALA A 1073 −21.261 69.675 20.620 1.00 68.05 C ATOM2172 N ALA A 1074 −20.797 67.584 22.890 1.00 69.54 N ATOM 2173 CA ALA A1074 −21.085 67.096 24.244 1.00 71.93 C ATOM 2174 C ALA A 1074 −19.79566.903 25.028 1.00 72.36 C ATOM 2175 O ALA A 1074 −19.684 67.378 26.1491.00 77.33 O ATOM 2176 CB ALA A 1074 −21.851 65.807 24.193 1.00 71.13 CATOM 2177 N VAL A 1075 −18.836 66.207 24.415 1.00 72.46 N ATOM 2178 CAVAL A 1075 −17.511 65.973 24.995 1.00 74.25 C ATOM 2179 C VAL A 1075−16.752 67.262 25.348 1.00 74.70 C ATOM 2180 O VAL A 1075 −16.163 67.34526.429 1.00 74.40 O ATOM 2181 CB VAL A 1075 −16.648 65.102 24.073 1.0074.74 C ATOM 2182 CG1 VAL A 1075 −15.208 65.076 24.539 1.00 75.33 C ATOM2183 CG2 VAL A 1075 −17.205 63.696 24.033 1.00 76.41 C ATOM 2184 N ARG A1076 −16.768 68.246 24.455 1.00 72.53 N ATOM 2185 CA ARG A 1076 −16.12469.546 24.726 1.00 74.21 C ATOM 2186 C ARG A 1076 −16.766 70.230 25.9111.00 74.53 C ATOM 2187 O ARG A 1076 −16.070 70.838 26.735 1.00 74.38 OATOM 2188 CB ARG A 1076 −16.204 70.487 23.533 1.00 70.53 C ATOM 2189 CGARG A 1076 −15.356 70.070 22.367 1.00 76.54 C ATOM 2190 CD ARG A 1076−15.465 71.070 21.205 1.00 82.85 C ATOM 2191 NE ARG A 1076 −15.06570.430 19.951 1.00 91.84 N ATOM 2192 CZ ARG A 1076 −13.802 70.183 19.5691.00 96.99 C ATOM 2193 NH1 ARG A 1076 −12.746 70.517 20.334 1.00 97.76 NATOM 2194 NH2 ARG A 1076 −13.580 69.588 18.393 1.00 103.37 N ATOM 2195 NGLY A 1077 −18.091 70.120 25.977 1.00 72.90 N ATOM 2196 CA GLY A 1077−18.865 70.656 27.059 1.00 73.02 C ATOM 2197 C GLY A 1077 −18.543 69.99528.392 1.00 72.32 C ATOM 2198 O GLY A 1077 −18.322 70.690 29.387 1.0073.12 O ATOM 2199 N ILE A 1078 −18.523 68.661 28.399 1.00 72.41 N ATOM2200 CA ILE A 1078 −18.104 67.858 29.564 1.00 73.34 C ATOM 2201 C ILE A1078 −16.739 68.293 30.083 1.00 71.54 C ATOM 2202 O ILE A 1078 −16.57768.522 31.280 1.00 76.16 O ATOM 2203 CB ILE A 1078 −18.044 66.363 29.2101.00 74.38 C ATOM 2204 CG1 ILE A 1078 −19.453 65.757 29.198 1.00 79.15 CATOM 2205 CG2 ILE A 1078 −17.205 65.616 30.174 1.00 64.66 C ATOM 2206CD1 ILE A 1078 −19.514 64.385 28.554 1.00 75.65 C ATOM 2207 N LEU A 1079−15.775 68.407 29.177 1.00 72.22 N ATOM 2208 CA LEU A 1079 −14.40768.819 29.512 1.00 73.11 C ATOM 2209 C LEU A 1079 −14.278 70.275 30.0011.00 73.92 C ATOM 2210 O LEU A 1079 −13.281 70.613 30.627 1.00 78.75 OATOM 2211 CB LEU A 1079 −13.462 68.578 28.317 1.00 72.14 C ATOM 2212 CGLEU A 1079 −13.192 67.115 27.909 1.00 72.35 C ATOM 2213 CD1 LEU A 1079−12.301 67.074 26.679 1.00 59.40 C ATOM 2214 CD2 LEU A 1079 −12.54366.309 29.031 1.00 62.86 C ATOM 2215 N ARG A 1080 −15.269 71.118 29.7141.00 73.61 N ATOM 2216 CA ARG A 1080 −15.330 72.490 30.251 1.00 74.19 CATOM 2217 C ARG A 1080 −16.039 72.537 31.609 1.00 72.82 C ATOM 2218 OARG A 1080 −15.894 73.505 32.331 1.00 72.73 O ATOM 2219 CB ARG A 1080−16.061 73.439 29.301 1.00 74.26 C ATOM 2220 CG ARG A 1080 −15.23673.955 28.140 1.00 79.49 C ATOM 2221 CD ARG A 1080 −15.964 75.121 27.4601.00 79.38 C ATOM 2222 NE ARG A 1080 −17.260 74.691 26.922 1.00 88.39 NATOM 2223 CZ ARG A 1080 −17.535 74.387 25.636 1.00 87.86 C ATOM 2224 NH1ARG A 1080 −16.608 74.451 24.665 1.00 87.22 N ATOM 2225 NH2 ARG A 1080−18.777 74.009 25.314 1.00 81.94 N ATOM 2226 N ASN A 1081 −16.800 71.49531.946 1.00 72.20 N ATOM 2227 CA ASN A 1081 −17.553 71.464 33.183 1.0070.14 C ATOM 2228 C ASN A 1081 −16.680 70.903 34.293 1.00 70.99 C ATOM2229 O ASN A 1081 −16.295 69.733 34.252 1.00 70.87 O ATOM 2230 CB ASN A1081 −18.817 70.642 33.002 1.00 68.83 C ATOM 2231 CG ASN A 1081 −19.78970.813 34.137 1.00 74.52 C ATOM 2232 OD1 ASN A 1081 −19.521 70.40535.275 1.00 81.88 O ATOM 2233 ND2 ASN A 1081 −20.937 71.417 33.848 1.0065.69 N ATOM 2234 N ALA A 1082 −16.374 71.750 35.281 1.00 69.81 N ATOM2235 CA ALA A 1082 −15.539 71.386 36.459 1.00 71.34 C ATOM 2236 C ALA A1082 −15.942 70.101 37.203 1.00 70.68 C ATOM 2237 O ALA A 1082 −15.08169.426 37.748 1.00 77.40 O ATOM 2238 CB ALA A 1082 −15.508 72.546 37.4511.00 64.36 C ATOM 2239 N LYS A 1083 −17.233 69.784 37.213 1.00 68.55 NATOM 2240 CA LYS A 1083 −17.776 68.623 37.931 1.00 69.05 C ATOM 2241 CLYS A 1083 −17.798 67.337 37.093 1.00 72.30 C ATOM 2242 O LYS A 1083−17.732 66.243 37.649 1.00 76.42 O ATOM 2243 CB LYS A 1083 −19.19368.923 38.398 1.00 70.45 C ATOM 2244 CG LYS A 1083 −19.359 70.297 39.0631.00 75.92 C ATOM 2245 CD LYS A 1083 −20.720 70.447 39.698 1.00 81.44 CATOM 2246 CE LYS A 1083 −20.926 71.864 40.217 1.00 85.50 C ATOM 2247 NZLYS A 1083 −22.148 71.994 41.068 1.00 88.29 N ATOM 2248 N LEU A 1084−17.891 67.460 35.765 1.00 68.40 N ATOM 2249 CA LEU A 1084 −17.89866.286 34.894 1.00 70.90 C ATOM 2250 C LEU A 1084 −16.518 65.924 34.4021.00 70.17 C ATOM 2251 O LEU A 1084 −16.247 64.740 34.205 1.00 72.79 OATOM 2252 CB LEU A 1084 −18.843 66.497 33.694 1.00 68.64 C ATOM 2253 CGLEU A 1084 −20.304 66.801 34.051 1.00 71.12 C ATOM 2254 CD1 LEU A 1084−21.213 66.916 32.787 1.00 59.19 C ATOM 2255 CD2 LEU A 1084 −20.84765.747 35.010 1.00 56.68 C ATOM 2256 N LYS A 1085 −15.650 66.917 34.2051.00 68.34 N ATOM 2257 CA LYS A 1085 −14.316 66.663 33.652 1.00 68.63 CATOM 2258 C LYS A 1085 −13.538 65.559 34.383 1.00 68.76 C ATOM 2259 OLYS A 1085 −13.070 64.643 33.723 1.00 75.06 O ATOM 2260 CB LYS A 1085−13.482 67.951 33.565 1.00 67.89 C ATOM 2261 CG LYS A 1085 −12.11067.770 32.917 1.00 69.24 C ATOM 2262 CD LYS A 1085 −11.322 69.054 32.9251.00 72.42 C ATOM 2263 CE LYS A 1085 −10.040 68.905 32.138 1.00 77.84 CATOM 2264 NZ LYS A 1085 −9.131 67.881 32.745 1.00 85.14 N ATOM 2265 NPRO A 1086 −13.399 65.636 35.740 1.00 69.24 N ATOM 2266 CA PRO A 1086−12.637 64.586 36.440 1.00 69.18 C ATOM 2267 C PRO A 1086 −13.226 63.19036.261 1.00 68.97 C ATOM 2268 O PRO A 1086 −12.488 62.197 36.199 1.0069.99 O ATOM 2269 CB PRO A 1086 −12.771 64.966 37.923 1.00 67.72 C ATOM2270 CG PRO A 1086 −13.201 66.357 37.950 1.00 72.53 C ATOM 2271 CD PRO A1086 −13.918 66.646 36.679 1.00 66.17 C ATOM 2272 N VAL A 1087 −14.54963.136 36.184 1.00 68.84 N ATOM 2273 CA VAL A 1087 −15.265 61.885 36.0921.00 72.28 C ATOM 2274 C VAL A 1087 −14.985 61.291 34.743 1.00 73.91 CATOM 2275 O VAL A 1087 −14.480 60.176 34.672 1.00 77.11 O ATOM 2276 CBVAL A 1087 −16.769 62.062 36.305 1.00 72.36 C ATOM 2277 CG1 VAL A 1087−17.431 60.720 36.368 1.00 78.50 C ATOM 2278 CG2 VAL A 1087 −17.03262.832 37.602 1.00 75.48 C ATOM 2279 N TYR A 1088 −15.310 62.047 33.6911.00 76.42 N ATOM 2280 CA TYR A 1088 −15.087 61.660 32.290 1.00 74.22 CATOM 2281 C TYR A 1088 −13.655 61.241 32.054 1.00 73.79 C ATOM 2282 OTYR A 1088 −13.414 60.184 31.502 1.00 71.26 O ATOM 2283 CB TYR A 1088−15.420 62.840 31.382 1.00 76.80 C ATOM 2284 CG TYR A 1088 −15.34562.536 29.909 1.00 78.30 C ATOM 2285 CD1 TYR A 1088 −16.447 61.99429.225 1.00 82.88 C ATOM 2286 CD2 TYR A 1088 −14.181 62.787 29.185 1.0082.17 C ATOM 2287 CE1 TYR A 1088 −16.368 61.712 27.838 1.00 78.90 C ATOM2288 CE2 TYR A 1088 −14.102 62.511 27.823 1.00 73.71 C ATOM 2289 CZ TYRA 1088 −15.199 61.976 27.162 1.00 71.96 C ATOM 2290 OH TYR A 1088−15.099 61.705 25.804 1.00 90.19 O ATOM 2291 N ASP A 1089 −12.708 62.08232.483 1.00 73.28 N ATOM 2292 CA ASP A 1089 −11.262 61.780 32.399 1.0072.57 C ATOM 2293 C ASP A 1089 −10.880 60.403 32.936 1.00 71.20 C ATOM2294 O ASP A 1089 −9.987 59.755 32.407 1.00 70.55 O ATOM 2295 CB ASP A1089 −10.438 62.802 33.208 1.00 72.85 C ATOM 2296 CG ASP A 1089 −10.16264.118 32.468 1.00 79.93 C ATOM 2297 OD1 ASP A 1089 −10.551 64.28731.274 1.00 78.98 O ATOM 2298 OD2 ASP A 1089 −9.528 65.004 33.120 1.0069.59 O ATOM 2299 N SER A 1090 −11.562 59.969 33.993 1.00 72.25 N ATOM2300 CA SER A 1090 −11.244 58.711 34.670 1.00 71.13 C ATOM 2301 C SER A1090 −11.893 57.495 34.027 1.00 69.01 C ATOM 2302 O SER A 1090 −11.41156.383 34.199 1.00 69.79 O ATOM 2303 CB SER A 1090 −11.684 58.791 36.1241.00 67.58 C ATOM 2304 OG SER A 1090 −13.085 58.665 36.232 1.00 65.84 OATOM 2305 N LEU A 1091 −12.979 57.703 33.293 1.00 72.26 N ATOM 2306 CALEU A 1091 −13.727 56.593 32.729 1.00 70.30 C ATOM 2307 C LEU A 1091−13.092 56.023 31.462 1.00 73.59 C ATOM 2308 O LEU A 1091 −12.420 56.71530.705 1.00 75.14 O ATOM 2309 CB LEU A 1091 −15.172 57.014 32.457 1.0073.21 C ATOM 2310 CG LEU A 1091 −16.060 57.298 33.687 1.00 69.70 C ATOM2311 CD1 LEU A 1091 −17.374 57.869 33.211 1.00 74.59 C ATOM 2312 CD2 LEUA 1091 −16.287 56.051 34.482 1.00 61.23 C ATOM 2313 N ASP A 1092 −13.32454.735 31.257 1.00 73.36 N ATOM 2314 CA ASP A 1092 −12.955 54.046 30.0171.00 74.54 C ATOM 2315 C ASP A 1092 −13.910 54.480 28.927 1.00 72.07 CATOM 2316 O ASP A 1092 −14.938 55.088 29.230 1.00 69.09 O ATOM 2317 CBASP A 1092 −13.093 52.547 30.180 1.00 74.48 C ATOM 2318 CG ASP A 1092−14.502 52.157 30.522 1.00 82.40 C ATOM 2319 OD1 ASP A 1092 −15.39352.059 29.643 1.00 78.10 O ATOM 2320 OD2 ASP A 1092 −14.702 51.95631.702 1.00 93.39 O ATOM 2321 N ALA A 1093 −13.561 54.156 27.677 1.0071.26 N ATOM 2322 CA ALA A 1093 −14.332 54.530 26.455 1.00 72.91 C ATOM2323 C ALA A 1093 −15.817 54.185 26.482 1.00 72.49 C ATOM 2324 O ALA A1093 −16.642 55.016 26.143 1.00 77.77 O ATOM 2325 CB ALA A 1093 −13.68253.884 25.213 1.00 68.92 C ATOM 2326 N VAL A 1094 −16.152 52.959 26.8821.00 74.25 N ATOM 2327 CA VAL A 1094 −17.566 52.517 26.970 1.00 76.09 CATOM 2328 C VAL A 1094 −18.388 53.297 28.021 1.00 74.99 C ATOM 2329 OVAL A 1094 −19.550 53.686 27.760 1.00 74.98 O ATOM 2330 CB VAL A 1094−17.680 51.014 27.268 1.00 77.56 C ATOM 2331 CG1 VAL A 1094 −19.12450.634 27.518 1.00 80.02 C ATOM 2332 CG2 VAL A 1094 −17.091 50.19226.098 1.00 77.82 C ATOM 2333 N ARG A 1095 −17.804 53.527 29.192 1.0074.55 N ATOM 2334 CA ARG A 1095 −18.502 54.306 30.245 1.00 72.87 C ATOM2335 C ARG A 1095 −18.564 55.792 29.877 1.00 70.86 C ATOM 2336 O ARG A1095 −19.529 56.471 30.224 1.00 70.60 O ATOM 2337 CB ARG A 1095 −17.85554.090 31.598 1.00 74.90 C ATOM 2338 CG ARG A 1095 −17.944 52.648 32.0491.00 74.88 C ATOM 2339 CD ARG A 1095 −17.456 52.519 33.465 1.00 76.13 CATOM 2340 NE ARG A 1095 −17.765 51.222 34.039 1.00 68.67 N ATOM 2341 CZARG A 1095 −17.035 50.111 33.935 1.00 75.88 C ATOM 2342 NH1 ARG A 1095−15.900 50.077 33.261 1.00 77.10 N ATOM 2343 NH2 ARG A 1095 −17.45648.999 34.523 1.00 78.82 N ATOM 2344 N ARG A 1096 −17.550 56.304 29.1761.00 70.40 N ATOM 2345 CA ARG A 1096 −17.646 57.654 28.616 1.00 72.78 CATOM 2346 C ARG A 1096 −18.895 57.781 27.683 1.00 75.95 C ATOM 2347 OARG A 1096 −19.603 58.790 27.741 1.00 76.20 O ATOM 2348 CB ARG A 1096−16.382 58.032 27.849 1.00 74.96 C ATOM 2349 CG ARG A 1096 −15.21258.381 28.729 1.00 76.80 C ATOM 2350 CD ARG A 1096 −13.987 58.699 27.8881.00 73.69 C ATOM 2351 NE ARG A 1096 −12.821 58.877 28.724 1.00 75.05 NATOM 2352 CZ ARG A 1096 −11.617 59.276 28.334 1.00 76.09 C ATOM 2353 NH1ARG A 1096 −11.358 59.568 27.062 1.00 79.66 N ATOM 2354 NH2 ARG A 1096−10.642 59.388 29.244 1.00 73.76 N ATOM 2355 N ALA A 1097 −19.154 56.76726.842 1.00 74.52 N ATOM 2356 CA ALA A 1097 −20.381 56.762 25.993 1.0075.40 C ATOM 2357 C ALA A 1097 −21.606 56.787 26.899 1.00 74.68 C ATOM2358 O ALA A 1097 −22.596 57.444 26.594 1.00 75.19 O ATOM 2359 CB ALA A1097 −20.428 55.556 25.084 1.00 65.57 C ATOM 2360 N ALA A 1098 −21.53856.068 28.019 1.00 77.29 N ATOM 2361 CA ALA A 1098 −22.627 56.110 29.0041.00 77.63 C ATOM 2362 C ALA A 1098 −22.855 57.549 29.425 1.00 75.46 CATOM 2363 O ALA A 1098 −23.953 58.037 29.306 1.00 77.90 O ATOM 2364 CBALA A 1098 −22.319 55.252 30.201 1.00 72.80 C ATOM 2365 N LEU A 1099−21.808 58.215 29.906 1.00 74.27 N ATOM 2366 CA LEU A 1099 −21.93559.610 30.389 1.00 73.67 C ATOM 2367 C LEU A 1099 −22.493 60.542 29.3291.00 73.73 C ATOM 2368 O LEU A 1099 −23.412 61.328 29.602 1.00 73.92 OATOM 2369 CB LEU A 1099 −20.590 60.144 30.886 1.00 76.16 C ATOM 2370 CGLEU A 1099 −20.612 61.381 31.794 1.00 76.75 C ATOM 2371 CD1 LEU A 1099−21.495 61.167 33.000 1.00 72.22 C ATOM 2372 CD2 LEU A 1099 −19.18761.741 32.236 1.00 66.69 C ATOM 2373 N ILE A 1100 −21.941 60.454 28.1151.00 72.03 N ATOM 2374 CA ILE A 1100 −22.430 61.252 26.986 1.00 70.39 CATOM 2375 C ILE A 1100 −23.908 60.998 26.758 1.00 69.34 C ATOM 2376 OILE A 1100 −24.674 61.941 26.552 1.00 70.67 O ATOM 2377 CB ILE A 1100−21.672 60.964 25.677 1.00 74.51 C ATOM 2378 CG1 ILE A 1100 −20.20861.411 25.789 1.00 74.12 C ATOM 2379 CG2 ILE A 1100 −22.368 61.68424.523 1.00 66.55 C ATOM 2380 CD1 ILE A 1100 −19.273 60.643 24.910 1.0071.96 C ATOM 2381 N ASN A 1101 −24.305 59.727 26.798 1.00 69.64 N ATOM2382 CA ASN A 1101 −25.716 59.340 26.656 1.00 71.15 C ATOM 2383 C ASN A1101 −26.587 60.154 27.596 1.00 73.26 C ATOM 2384 O ASN A 1101 −27.60660.720 27.158 1.00 73.01 O ATOM 2385 CB ASN A 1101 −25.859 57.819 26.8801.00 74.91 C ATOM 2386 CG ASN A 1101 −27.242 57.286 26.621 1.00 72.41 CATOM 2387 OD1 ASN A 1101 −28.218 57.895 26.974 1.00 68.21 O ATOM 2388ND2 ASN A 1101 −27.321 56.110 25.995 1.00 66.91 N ATOM 2389 N MET A 1102−26.191 60.217 28.876 1.00 73.18 N ATOM 2390 CA MET A 1102 −26.92260.986 29.894 1.00 73.53 C ATOM 2391 C MET A 1102 −26.897 62.472 29.5791.00 72.72 C ATOM 2392 O MET A 1102 −27.909 63.130 29.719 1.00 75.34 OATOM 2393 CB MET A 1102 −26.341 60.788 31.315 1.00 69.92 C ATOM 2394 CGMET A 1102 −26.311 59.343 31.846 1.00 71.61 C ATOM 2395 SD MET A 1102−25.639 59.205 33.558 1.00 75.01 S ATOM 2396 CE MET A 1102 −26.90360.133 34.436 1.00 72.22 C ATOM 2397 N VAL A 1103 −25.743 62.997 29.1601.00 73.08 N ATOM 2398 CA VAL A 1103 −25.650 64.430 28.774 1.00 75.10 CATOM 2399 C VAL A 1103 −26.605 64.736 27.585 1.00 74.50 C ATOM 2400 OVAL A 1103 −27.280 65.769 27.587 1.00 74.33 O ATOM 2401 CB VAL A 1103−24.203 64.859 28.445 1.00 74.63 C ATOM 2402 CG1 VAL A 1103 −24.18166.214 27.773 1.00 74.66 C ATOM 2403 CG2 VAL A 1103 −23.345 64.89229.715 1.00 73.28 C ATOM 2404 N PHE A 1104 −26.648 63.838 26.598 1.0072.50 N ATOM 2405 CA PHE A 1104 −27.641 63.937 25.507 1.00 77.14 C ATOM2406 C PHE A 1104 −29.050 64.133 26.087 1.00 77.59 C ATOM 2407 O PHE A1104 −29.746 65.023 25.684 1.00 78.04 O ATOM 2408 CB PHE A 1104 −27.61562.712 24.557 1.00 78.31 C ATOM 2409 CG PHE A 1104 −26.870 62.933 23.2591.00 81.99 C ATOM 2410 CD1 PHE A 1104 −25.489 63.029 23.238 1.00 78.79 CATOM 2411 CD2 PHE A 1104 −27.580 63.037 22.027 1.00 90.20 C ATOM 2412CE1 PHE A 1104 −24.800 63.227 22.021 1.00 77.16 C ATOM 2413 CE2 PHE A1104 −26.910 63.237 20.821 1.00 84.00 C ATOM 2414 CZ PHE A 1104 −25.50963.332 20.823 1.00 86.09 C ATOM 2415 N GLN A 1105 −29.446 63.296 27.0391.00 76.01 N ATOM 2416 CA GLN A 1105 −30.818 63.335 27.575 1.00 75.44 CATOM 2417 C GLN A 1105 −31.159 64.480 28.544 1.00 74.63 C ATOM 2418 OGLN A 1105 −32.249 65.043 28.455 1.00 75.26 O ATOM 2419 CB GLN A 1105−31.100 62.012 28.270 1.00 77.85 C ATOM 2420 CG GLN A 1105 −32.56761.757 28.615 1.00 75.54 C ATOM 2421 CD GLN A 1105 −32.781 60.378 29.1681.00 75.71 C ATOM 2422 OE1 GLN A 1105 −31.834 59.620 29.340 1.00 71.29 OATOM 2423 NE2 GLN A 1105 −34.025 60.039 29.450 1.00 70.96 N ATOM 2424 NMET A 1106 −30.250 64.819 29.456 1.00 76.62 N ATOM 2425 CA MET A 1106−30.529 65.838 30.524 1.00 77.20 C ATOM 2426 C MET A 1106 −29.666 67.10030.498 1.00 75.43 C ATOM 2427 O MET A 1106 −29.901 68.010 31.296 1.0078.05 O ATOM 2428 CB MET A 1106 −30.319 65.216 31.902 1.00 79.39 C ATOM2429 CG MET A 1106 −30.886 63.832 32.124 1.00 85.85 C ATOM 2430 SD MET A1106 −30.091 63.064 33.542 1.00 85.72 S ATOM 2431 CE MET A 1106 −29.93361.419 32.917 1.00 81.10 C ATOM 2432 N GLY A 1107 −28.675 67.166 29.6091.00 75.52 N ATOM 2433 CA GLY A 1107 −27.748 68.294 29.560 1.00 76.26 CATOM 2434 C GLY A 1107 −26.702 68.166 30.651 1.00 78.60 C ATOM 2435 OGLY A 1107 −26.827 67.330 31.555 1.00 78.11 O ATOM 2436 N GLU A 1108−25.666 68.993 30.573 1.00 79.86 N ATOM 2437 CA GLU A 1108 −24.55668.931 31.548 1.00 82.66 C ATOM 2438 C GLU A 1108 −24.966 69.224 32.9661.00 82.97 C ATOM 2439 O GLU A 1108 −24.495 68.560 33.891 1.00 86.76 OATOM 2440 CB GLU A 1108 −23.454 69.901 31.185 1.00 82.78 C ATOM 2441 CGGLU A 1108 −22.714 69.512 29.969 1.00 86.49 C ATOM 2442 CD GLU A 1108−21.722 70.556 29.554 1.00 90.68 C ATOM 2443 OE1 GLU A 1108 −21.14871.271 30.409 1.00 90.93 O ATOM 2444 OE2 GLU A 1108 −21.523 70.65928.348 1.00 103.73 O ATOM 2445 N THR A 1109 −25.837 70.214 33.132 1.0082.33 N ATOM 2446 CA THR A 1109 −26.324 70.597 34.451 1.00 80.83 C ATOM2447 C THR A 1109 −27.061 69.455 35.112 1.00 79.03 C ATOM 2448 O THR A1109 −26.755 69.098 36.247 1.00 84.70 O ATOM 2449 CB THR A 1109 −27.27071.802 34.386 1.00 79.62 C ATOM 2450 OG1 THR A 1109 −26.591 72.90733.776 1.00 80.39 O ATOM 2451 CG2 THR A 1109 −27.731 72.196 35.790 1.0081.01 C ATOM 2452 N GLY A 1110 −28.028 68.889 34.395 1.00 78.14 N ATOM2453 CA GLY A 1110 −28.794 67.749 34.888 1.00 77.59 C ATOM 2454 C GLY A1110 −27.893 66.637 35.399 1.00 76.91 C ATOM 2455 O GLY A 1110 −28.14766.069 36.450 1.00 77.84 O ATOM 2456 N VAL A 1111 −26.838 66.348 34.6391.00 74.81 N ATOM 2457 CA VAL A 1111 −25.901 65.274 34.952 1.00 73.76 CATOM 2458 C VAL A 1111 −24.901 65.663 36.063 1.00 74.74 C ATOM 2459 OVAL A 1111 −24.475 64.809 36.863 1.00 70.56 O ATOM 2460 CB VAL A 1111−25.094 64.844 33.715 1.00 68.49 C ATOM 2461 CG1 VAL A 1111 −24.17363.695 34.081 1.00 64.99 C ATOM 2462 CG2 VAL A 1111 −26.023 64.43832.582 1.00 68.09 C ATOM 2463 N ALA A 1112 −24.531 66.949 36.096 1.0074.29 N ATOM 2464 CA ALA A 1112 −23.631 67.491 37.119 1.00 77.63 C ATOM2465 C ALA A 1112 −24.255 67.423 38.496 1.00 77.53 C ATOM 2466 O ALA A1112 −23.542 67.410 39.483 1.00 82.24 O ATOM 2467 CB ALA A 1112 −23.25668.931 36.798 1.00 77.58 C ATOM 2468 N GLY A 1113 −25.591 67.381 38.5531.00 79.76 N ATOM 2469 CA GLY A 1113 −26.323 67.270 39.808 1.00 77.64 CATOM 2470 C GLY A 1113 −26.267 65.900 40.461 1.00 78.25 C ATOM 2471 OGLY A 1113 −26.687 65.780 41.588 1.00 76.50 O ATOM 2472 N PHE A 1114−25.757 64.861 39.774 1.00 77.88 N ATOM 2473 CA PHE A 1114 −25.58663.533 40.383 1.00 76.03 C ATOM 2474 C PHE A 1114 −24.276 63.500 41.1791.00 75.36 C ATOM 2475 O PHE A 1114 −23.399 62.693 40.887 1.00 73.89 OATOM 2476 CB PHE A 1114 −25.559 62.437 39.310 1.00 81.97 C ATOM 2477 CGPHE A 1114 −26.845 62.247 38.594 1.00 84.39 C ATOM 2478 CD1 PHE A 1114−27.906 61.609 39.202 1.00 93.33 C ATOM 2479 CD2 PHE A 1114 −27.00162.696 37.310 1.00 90.99 C ATOM 2480 CE1 PHE A 1114 −29.108 61.43338.521 1.00 91.37 C ATOM 2481 CE2 PHE A 1114 −28.187 62.526 36.627 1.0091.29 C ATOM 2482 CZ PHE A 1114 −29.244 61.894 37.229 1.00 89.69 C ATOM2483 N THR A 1115 −24.139 64.364 42.186 1.00 70.77 N ATOM 2484 CA THR A1115 −22.860 64.550 42.845 1.00 71.53 C ATOM 2485 C THR A 1115 −22.29263.256 43.420 1.00 72.52 C ATOM 2486 O THR A 1115 −21.124 62.964 43.2061.00 74.92 O ATOM 2487 CB THR A 1115 −22.950 65.613 43.957 1.00 74.18 CATOM 2488 OG1 THR A 1115 −23.638 66.761 43.454 1.00 81.29 O ATOM 2489CG2 THR A 1115 −21.562 66.027 44.419 1.00 65.46 C ATOM 2490 N ASN A 1116−23.112 62.494 44.138 1.00 73.53 N ATOM 2491 CA ASN A 1116 −22.64461.260 44.779 1.00 74.43 C ATOM 2492 C ASN A 1116 −22.327 60.176 43.7531.00 74.36 C ATOM 2493 O ASN A 1116 −21.286 59.523 43.854 1.00 73.98 OATOM 2494 CB ASN A 1116 −23.652 60.723 45.803 1.00 76.08 C ATOM 2495 CGASN A 1116 −23.916 61.701 46.958 1.00 81.08 C ATOM 2496 OD1 ASN A 1116−22.994 62.125 47.647 1.00 76.24 O ATOM 2497 ND2 ASN A 1116 −25.18162.050 47.163 1.00 82.05 N ATOM 2498 N SER A 1117 −23.218 59.986 42.7761.00 71.66 N ATOM 2499 CA SER A 1117 −22.990 59.014 41.697 1.00 71.35 CATOM 2500 C SER A 1117 −21.736 59.307 40.895 1.00 72.60 C ATOM 2501 OSER A 1117 −21.010 58.386 40.567 1.00 73.79 O ATOM 2502 CB SER A 1117−24.170 58.962 40.748 1.00 74.52 C ATOM 2503 OG SER A 1117 −25.27158.329 41.351 1.00 74.10 O ATOM 2504 N LEU A 1118 −21.484 60.580 40.5861.00 74.22 N ATOM 2505 CA LEU A 1118 −20.253 60.997 39.863 1.00 74.12 CATOM 2506 C LEU A 1118 −18.961 60.660 40.618 1.00 73.96 C ATOM 2507 OLEU A 1118 −17.984 60.195 40.025 1.00 78.17 O ATOM 2508 CB LEU A 1118−20.299 62.503 39.566 1.00 74.92 C ATOM 2509 CG LEU A 1118 −21.33762.937 38.500 1.00 77.06 C ATOM 2510 CD1 LEU A 1118 −21.516 64.48438.518 1.00 79.91 C ATOM 2511 CD2 LEU A 1118 −20.950 62.456 37.120 1.0079.15 C ATOM 2512 N ARG A 1119 −18.972 60.901 41.917 1.00 73.81 N ATOM2513 CA ARG A 1119 −17.857 60.571 42.806 1.00 76.37 C ATOM 2514 C ARG A1119 −17.586 59.069 42.837 1.00 73.72 C ATOM 2515 O ARG A 1119 −16.43958.633 42.760 1.00 71.54 O ATOM 2516 CB ARG A 1119 −18.179 61.041 44.2291.00 72.72 C ATOM 2517 CG ARG A 1119 −17.067 60.873 45.241 1.00 81.26 CATOM 2518 CD ARG A 1119 −17.608 60.978 46.712 1.00 88.03 C ATOM 2519 NEARG A 1119 −18.622 62.027 46.876 1.00 97.80 N ATOM 2520 CZ ARG A 1119−18.387 63.350 46.852 1.00 107.35 C ATOM 2521 NH1 ARG A 1119 −17.14963.849 46.666 1.00 110.91 N ATOM 2522 NH2 ARG A 1119 −19.407 64.20747.016 1.00 108.55 N ATOM 2523 N MET A 1120 −18.653 58.296 42.958 1.0072.40 N ATOM 2524 CA MET A 1120 −18.554 56.843 42.948 1.00 75.44 C ATOM2525 C MET A 1120 −18.059 56.323 41.610 1.00 72.59 C ATOM 2526 O MET A1120 −17.199 55.455 41.580 1.00 74.45 O ATOM 2527 CB MET A 1120 −19.88456.228 43.338 1.00 74.73 C ATOM 2528 CG MET A 1120 −20.116 56.407 44.8611.00 79.31 C ATOM 2529 SD MET A 1120 −21.698 55.810 45.341 1.00 86.73 SATOM 2530 CE MET A 1120 −22.843 56.943 44.571 1.00 82.05 C ATOM 2531 NLEU A 1121 −18.592 56.851 40.516 1.00 70.51 N ATOM 2532 CA LEU A 1121−18.067 56.495 39.211 1.00 73.43 C ATOM 2533 C LEU A 1121 −16.581 56.81639.149 1.00 71.65 C ATOM 2534 O LEU A 1121 −15.810 55.970 38.728 1.0074.87 O ATOM 2535 CB LEU A 1121 −18.802 57.213 38.086 1.00 67.49 C ATOM2536 CG LEU A 1121 −20.247 56.734 37.877 1.00 72.80 C ATOM 2537 CD1 LEUA 1121 −20.990 57.724 36.968 1.00 65.17 C ATOM 2538 CD2 LEU A 1121−20.343 55.241 37.364 1.00 62.04 C ATOM 2539 N GLN A 1122 −16.186 58.02039.565 1.00 69.51 N ATOM 2540 CA GLN A 1122 −14.760 58.419 39.551 1.0069.43 C ATOM 2541 C GLN A 1122 −13.852 57.422 40.302 1.00 69.96 C ATOM2542 O GLN A 1122 −12.726 57.188 39.889 1.00 72.67 O ATOM 2543 CB GLN A1122 −14.602 59.828 40.123 1.00 69.75 C ATOM 2544 CG GLN A 1122 −13.20360.432 39.975 1.00 73.87 C ATOM 2545 CD GLN A 1122 −13.125 61.883 40.4431.00 79.31 C ATOM 2546 OE1 GLN A 1122 −14.139 62.505 40.758 1.00 88.60 OATOM 2547 NE2 GLN A 1122 −11.907 62.426 40.490 1.00 84.85 N ATOM 2548 NGLN A 1123 −14.364 56.850 41.395 1.00 66.33 N ATOM 2549 CA GLN A 1123−13.659 55.833 42.182 1.00 71.40 C ATOM 2550 C GLN A 1123 −13.857 54.37341.688 1.00 70.37 C ATOM 2551 O GLN A 1123 −13.323 53.450 42.300 1.0068.04 O ATOM 2552 CB GLN A 1123 −14.083 55.958 43.662 1.00 66.15 C ATOM2553 CG GLN A 1123 −13.701 57.318 44.267 1.00 65.21 C ATOM 2554 CD GLN A1123 −14.267 57.566 45.652 1.00 69.71 C ATOM 2555 OE1 GLN A 1123 −15.29457.018 46.035 1.00 77.32 O ATOM 2556 NE2 GLN A 1123 −13.585 58.40446.411 1.00 63.29 N ATOM 2557 N LYS A 1124 −14.609 54.174 40.604 1.0069.50 N ATOM 2558 CA LYS A 1124 −14.896 52.830 40.066 1.00 74.09 C ATOM2559 C LYS A 1124 −15.719 51.959 41.048 1.00 75.70 C ATOM 2560 O LYS A1124 −15.562 50.733 41.084 1.00 75.06 O ATOM 2561 CB LYS A 1124 −13.60352.089 39.688 1.00 79.02 C ATOM 2562 CG LYS A 1124 −12.571 52.892 38.8921.00 83.65 C ATOM 2563 CD LYS A 1124 −12.682 52.701 37.399 1.00 89.63 CATOM 2564 CE LYS A 1124 −11.978 53.758 36.540 1.00 92.96 C ATOM 2565 NZLYS A 1124 −12.833 55.011 36.416 1.00 104.91 N ATOM 2566 N ARG A 1125−16.586 52.609 41.828 1.00 74.41 N ATOM 2567 CA ARG A 1125 −17.48951.958 42.758 1.00 73.39 C ATOM 2568 C ARG A 1125 −18.787 51.862 41.9751.00 72.92 C ATOM 2569 O ARG A 1125 −19.755 52.569 42.238 1.00 75.78 OATOM 2570 CB ARG A 1125 −17.625 52.765 44.049 1.00 72.14 C ATOM 2571 CGARG A 1125 −16.304 52.959 44.776 1.00 74.10 C ATOM 2572 CD ARG A 1125−16.448 53.860 45.974 1.00 76.04 C ATOM 2573 NE ARG A 1125 −17.14453.226 47.096 1.00 83.94 N ATOM 2574 CZ ARG A 1125 −17.567 53.859 48.2031.00 87.62 C ATOM 2575 NH1 ARG A 1125 −17.373 55.179 48.365 1.00 91.97 NATOM 2576 NH2 ARG A 1125 −18.195 53.171 49.172 1.00 84.34 N ATOM 2577 NTRP A 1126 −18.778 50.963 41.005 1.00 74.20 N ATOM 2578 CA TRP A 1126−19.839 50.847 39.981 1.00 72.97 C ATOM 2579 C TRP A 1126 −21.220 50.50740.541 1.00 71.90 C ATOM 2580 O TRP A 1126 −22.203 51.187 40.232 1.0067.72 O ATOM 2581 CB TRP A 1126 −19.473 49.781 38.959 1.00 69.76 C ATOM2582 CG TRP A 1126 −18.124 49.894 38.335 1.00 70.87 C ATOM 2583 CD1 TRPA 1126 −17.183 48.917 38.255 1.00 66.79 C ATOM 2584 CD2 TRP A 1126−17.564 51.047 37.699 1.00 70.96 C ATOM 2585 NE1 TRP A 1126 −16.06849.386 37.610 1.00 69.94 N ATOM 2586 CE2 TRP A 1126 −16.280 50.69237.260 1.00 70.31 C ATOM 2587 CE3 TRP A 1126 −18.026 52.351 37.457 1.0071.68 C ATOM 2588 CZ2 TRP A 1126 −15.460 51.576 36.600 1.00 74.24 C ATOM2589 CZ3 TRP A 1126 −17.205 53.235 36.801 1.00 71.20 C ATOM 2590 CH2 TRPA 1126 −15.939 52.850 36.379 1.00 73.75 C ATOM 2591 N ASP A 1127 −21.27349.458 41.357 1.00 72.11 N ATOM 2592 CA ASP A 1127 −22.537 49.012 41.9941.00 74.28 C ATOM 2593 C ASP A 1127 −23.168 50.100 42.830 1.00 72.96 CATOM 2594 O ASP A 1127 −24.377 50.283 42.781 1.00 77.35 O ATOM 2595 CBASP A 1127 −22.326 47.768 42.874 1.00 73.85 C ATOM 2596 CG ASP A 1127−22.177 46.475 42.065 1.00 80.36 C ATOM 2597 OD1 ASP A 1127 −22.03446.530 40.836 1.00 81.11 O ATOM 2598 OD2 ASP A 1127 −22.204 45.38642.681 1.00 97.24 O ATOM 2599 N GLU A 1128 −22.349 50.813 43.586 1.0070.74 N ATOM 2600 CA GLU A 1128 −22.840 51.886 44.452 1.00 72.33 C ATOM2601 C GLU A 1128 −23.299 53.089 43.646 1.00 70.63 C ATOM 2602 O GLU A1128 −24.276 53.740 44.017 1.00 70.95 O ATOM 2603 CB GLU A 1128 −21.76452.293 45.420 1.00 70.41 C ATOM 2604 CG GLU A 1128 −21.384 51.186 46.3641.00 79.81 C ATOM 2605 CD GLU A 1128 −20.232 51.570 47.219 1.00 81.59 CATOM 2606 OE1 GLU A 1128 −20.445 52.476 48.059 1.00 90.24 O ATOM 2607OE2 GLU A 1128 −19.131 50.969 47.053 1.00 85.83 O ATOM 2608 N ALA A 1129−22.587 53.372 42.561 1.00 67.55 N ATOM 2609 CA ALA A 1129 −23.00054.375 41.574 1.00 71.98 C ATOM 2610 C ALA A 1129 −24.378 54.000 41.0581.00 70.13 C ATOM 2611 O ALA A 1129 −25.263 54.827 41.057 1.00 76.48 OATOM 2612 CB ALA A 1129 −21.989 54.462 40.410 1.00 65.05 C ATOM 2613 NALA A 1130 −24.534 52.742 40.634 1.00 72.74 N ATOM 2614 CA ALA A 1130−25.821 52.182 40.126 1.00 70.99 C ATOM 2615 C ALA A 1130 −26.956 52.24941.142 1.00 70.01 C ATOM 2616 O ALA A 1130 −28.122 52.487 40.776 1.0068.38 O ATOM 2617 CB ALA A 1130 −25.623 50.735 39.676 1.00 66.12 C ATOM2618 N VAL A 1131 −26.632 52.039 42.416 1.00 68.58 N ATOM 2619 CA VAL A1131 −27.663 52.124 43.473 1.00 67.85 C ATOM 2620 C VAL A 1131 −28.22253.535 43.503 1.00 69.11 C ATOM 2621 O VAL A 1131 −29.445 53.719 43.5511.00 67.62 O ATOM 2622 CB VAL A 1131 −27.149 51.685 44.851 1.00 69.41 CATOM 2623 CG1 VAL A 1131 −28.070 52.236 46.024 1.00 60.58 C ATOM 2624CG2 VAL A 1131 −27.016 50.147 44.886 1.00 64.52 C ATOM 2625 N ASN A 1132−27.323 54.517 43.466 1.00 71.57 N ATOM 2626 CA ASN A 1132 −27.70655.935 43.510 1.00 73.21 C ATOM 2627 C ASN A 1132 −28.408 56.412 42.2321.00 70.74 C ATOM 2628 O ASN A 1132 −29.422 57.104 42.307 1.00 68.21 OATOM 2629 CB ASN A 1132 −26.486 56.817 43.828 1.00 72.85 C ATOM 2630 CGASN A 1132 −26.112 56.776 45.296 1.00 78.49 C ATOM 2631 OD1 ASN A 1132−26.088 57.807 45.962 1.00 83.67 O ATOM 2632 ND2 ASN A 1132 −25.82255.588 45.808 1.00 84.32 N ATOM 2633 N LEU A 1133 −27.869 56.040 41.0761.00 69.80 N ATOM 2634 CA LEU A 1133 −28.465 56.421 39.798 1.00 72.25 CATOM 2635 C LEU A 1133 −29.883 55.859 39.633 1.00 73.77 C ATOM 2636 OLEU A 1133 −30.748 56.528 39.065 1.00 78.51 O ATOM 2637 CB LEU A 1133−27.569 55.971 38.637 1.00 74.37 C ATOM 2638 CG LEU A 1133 −26.18656.632 38.565 1.00 74.55 C ATOM 2639 CD1 LEU A 1133 −25.327 56.04537.436 1.00 81.95 C ATOM 2640 CD2 LEU A 1133 −26.336 58.139 38.408 1.0079.93 C ATOM 2641 N ALA A 1134 −30.121 54.639 40.132 1.00 72.16 N ATOM2642 CA ALA A 1134 −31.455 54.022 40.090 1.00 71.47 C ATOM 2643 C ALA A1134 −32.505 54.776 40.950 1.00 72.41 C ATOM 2644 O ALA A 1134 −33.70154.586 40.741 1.00 70.87 O ATOM 2645 CB ALA A 1134 −31.378 52.533 40.5111.00 68.95 C ATOM 2646 N LYS A 1135 −32.052 55.620 41.901 1.00 68.74 NATOM 2647 CA LYS A 1135 −32.946 56.436 42.773 1.00 72.99 C ATOM 2648 CLYS A 1135 −33.266 57.857 42.231 1.00 72.10 C ATOM 2649 O LYS A 1135−33.585 58.762 43.000 1.00 74.92 O ATOM 2650 CB LYS A 1135 −32.34356.536 44.199 1.00 69.32 C ATOM 2651 CG LYS A 1135 −32.402 55.253 45.0121.00 76.21 C ATOM 2652 CD LYS A 1135 −31.674 55.410 46.367 1.00 77.13 CATOM 2653 CE LYS A 1135 −31.950 54.245 47.310 1.00 77.91 C ATOM 2654 NZLYS A 1135 −30.938 54.134 48.434 1.00 77.31 N ATOM 2655 N SER A 1136−33.185 58.051 40.916 1.00 76.65 N ATOM 2656 CA SER A 1136 −33.40459.364 40.298 1.00 74.00 C ATOM 2657 C SER A 1136 −34.753 59.516 39.5941.00 76.99 C ATOM 2658 O SER A 1136 −35.464 58.536 39.320 1.00 79.35 OATOM 2659 CB SER A 1136 −32.289 59.635 39.304 1.00 75.82 C ATOM 2660 OGSER A 1136 −32.139 58.573 38.368 1.00 74.98 O ATOM 2661 N ARG A 1137−35.081 60.775 39.313 1.00 74.80 N ATOM 2662 CA ARG A 1137 −36.21661.141 38.485 1.00 72.91 C ATOM 2663 C ARG A 1137 −35.965 60.639 37.0431.00 71.62 C ATOM 2664 O ARG A 1137 −36.884 60.312 36.325 1.00 70.32 OATOM 2665 CB ARG A 1137 −36.395 62.661 38.504 1.00 71.58 C ATOM 2666 CGARG A 1137 −37.567 63.220 37.702 1.00 76.14 C ATOM 2667 CD ARG A 1137−38.896 62.715 38.231 1.00 84.99 C ATOM 2668 NE ARG A 1137 −40.03463.267 37.497 1.00 85.80 N ATOM 2669 CZ ARG A 1137 −41.305 62.894 37.6641.00 83.56 C ATOM 2670 NH1 ARG A 1137 −41.640 61.951 38.547 1.00 74.66 NATOM 2671 NH2 ARG A 1137 −42.263 63.469 36.933 1.00 88.55 N ATOM 2672 NTRP A 1138 −34.703 60.592 36.651 1.00 70.67 N ATOM 2673 CA TRP A 1138−34.289 60.023 35.392 1.00 72.64 C ATOM 2674 C TRP A 1138 −34.759 58.56935.248 1.00 74.44 C ATOM 2675 O TRP A 1138 −35.428 58.233 34.279 1.0074.91 O ATOM 2676 CB TRP A 1138 −32.778 60.118 35.298 1.00 72.63 C ATOM2677 CG TRP A 1138 −32.166 59.434 34.164 1.00 72.19 C ATOM 2678 CD1 TRPA 1138 −32.556 59.491 32.862 1.00 72.21 C ATOM 2679 CD2 TRP A 1138−31.024 58.574 34.207 1.00 68.92 C ATOM 2680 NE1 TRP A 1138 −31.72458.713 32.092 1.00 73.58 N ATOM 2681 CE2 TRP A 1138 −30.780 58.13932.887 1.00 67.95 C ATOM 2682 CE3 TRP A 1138 −30.190 58.125 35.223 1.0066.16 C ATOM 2683 CZ2 TRP A 1138 −29.749 57.293 32.570 1.00 69.41 C ATOM2684 CZ3 TRP A 1138 −29.142 57.262 34.893 1.00 71.01 C ATOM 2685 CH2 TRPA 1138 −28.934 56.863 33.600 1.00 71.24 C ATOM 2686 N TYR A 1139 −34.40757.727 36.212 1.00 73.44 N ATOM 2687 CA TYR A 1139 −34.828 56.318 36.1961.00 70.88 C ATOM 2688 C TYR A 1139 −36.337 56.170 36.397 1.00 71.38 CATOM 2689 O TYR A 1139 −36.940 55.218 35.922 1.00 72.03 O ATOM 2690 CBTYR A 1139 −34.064 55.516 37.258 1.00 74.06 C ATOM 2691 CG TYR A 1139−34.530 54.095 37.404 1.00 70.82 C ATOM 2692 CD1 TYR A 1139 −34.01253.101 36.601 1.00 76.39 C ATOM 2693 CD2 TYR A 1139 −35.496 53.74638.351 1.00 76.13 C ATOM 2694 CE1 TYR A 1139 −34.429 51.793 36.720 1.0073.02 C ATOM 2695 CE2 TYR A 1139 −35.923 52.436 38.481 1.00 79.81 C ATOM2696 CZ TYR A 1139 −35.376 51.461 37.651 1.00 77.76 C ATOM 2697 OH TYR A1139 −35.772 50.154 37.747 1.00 78.31 O ATOM 2698 N ASN A 1140 −36.94357.111 37.106 1.00 72.72 N ATOM 2699 CA ASN A 1140 −38.381 57.111 37.3121.00 71.50 C ATOM 2700 C ASN A 1140 −39.175 57.391 36.028 1.00 70.46 CATOM 2701 O ASN A 1140 −40.307 56.929 35.882 1.00 72.15 O ATOM 2702 CBASN A 1140 −38.755 58.172 38.338 1.00 68.60 C ATOM 2703 CG ASN A 1140−40.230 58.240 38.572 1.00 72.49 C ATOM 2704 OD1 ASN A 1140 −40.86459.264 38.335 1.00 72.97 O ATOM 2705 ND2 ASN A 1140 −40.801 57.13839.040 1.00 83.45 N ATOM 2706 N GLN A 1141 −38.573 58.145 35.111 1.0071.15 N ATOM 2707 CA GLN A 1141 −39.248 58.616 33.896 1.00 71.46 C ATOM2708 C GLN A 1141 −38.919 57.755 32.677 1.00 72.46 C ATOM 2709 O GLN A1141 −39.828 57.378 31.930 1.00 69.45 O ATOM 2710 CB GLN A 1141 −38.88760.091 33.663 1.00 71.54 C ATOM 2711 CG GLN A 1141 −39.493 61.040 34.6941.00 74.06 C ATOM 2712 CD GLN A 1141 −40.987 61.142 34.576 1.00 84.66 CATOM 2713 OE1 GLN A 1141 −41.726 60.867 35.539 1.00 72.30 O ATOM 2714NE2 GLN A 1141 −41.461 61.535 33.387 1.00 78.96 N ATOM 2715 N THR A 1142−37.633 57.454 32.490 1.00 69.68 N ATOM 2716 CA THR A 1142 −37.16756.582 31.409 1.00 71.24 C ATOM 2717 C THR A 1142 −36.399 55.382 31.9971.00 71.57 C ATOM 2718 O THR A 1142 −35.177 55.274 31.783 1.00 73.67 OATOM 2719 CB THR A 1142 −36.318 57.373 30.396 1.00 73.10 C ATOM 2720 OG1THR A 1142 −35.335 58.134 31.100 1.00 65.73 O ATOM 2721 CG2 THR A 1142−37.223 58.326 29.569 1.00 63.92 C ATOM 2722 N PRO A 1143 −37.120 54.47532.744 1.00 68.91 N ATOM 2723 CA PRO A 1143 −36.537 53.299 33.404 1.0067.95 C ATOM 2724 C PRO A 1143 −35.762 52.372 32.497 1.00 71.67 C ATOM2725 O PRO A 1143 −34.638 52.018 32.834 1.00 70.47 O ATOM 2726 CB PRO A1143 −37.760 52.553 33.937 1.00 70.48 C ATOM 2727 CG PRO A 1143 −38.92053.137 33.252 1.00 70.91 C ATOM 2728 CD PRO A 1143 −38.570 54.528 33.0011.00 68.98 C ATOM 2729 N ASN A 1144 −36.357 51.993 31.361 1.00 70.87 NATOM 2730 CA ASN A 1144 −35.706 51.083 30.410 1.00 71.14 C ATOM 2731 CASN A 1144 −34.382 51.613 29.946 1.00 72.08 C ATOM 2732 O ASN A 1144−33.369 50.921 30.071 1.00 71.77 O ATOM 2733 CB ASN A 1144 −36.58250.818 29.180 1.00 71.11 C ATOM 2734 CG ASN A 1144 −37.859 50.077 29.5151.00 74.27 C ATOM 2735 OD1 ASN A 1144 −37.919 49.323 30.480 1.00 76.06 OATOM 2736 ND2 ASN A 1144 −38.895 50.288 28.709 1.00 86.76 N ATOM 2737 NARG A 1145 −34.388 52.837 29.409 1.00 70.29 N ATOM 2738 CA ARG A 1145−33.147 53.500 28.979 1.00 69.78 C ATOM 2739 C ARG A 1145 −32.142 53.62730.132 1.00 70.79 C ATOM 2740 O ARG A 1145 −30.950 53.299 29.984 1.0071.02 O ATOM 2741 CB ARG A 1145 −33.450 54.880 28.462 1.00 69.40 C ATOM2742 CG ARG A 1145 −32.215 55.697 28.103 1.00 72.46 C ATOM 2743 CD ARG A1145 −32.619 56.971 27.368 1.00 71.18 C ATOM 2744 NE ARG A 1145 −31.44657.732 26.931 1.00 74.68 N ATOM 2745 CZ ARG A 1145 −31.488 58.840 26.1861.00 81.63 C ATOM 2746 NH1 ARG A 1145 −32.661 59.353 25.769 1.00 76.56 NATOM 2747 NH2 ARG A 1145 −30.354 59.453 25.844 1.00 80.74 N ATOM 2748 NALA A 1146 −32.632 54.105 31.272 1.00 68.56 N ATOM 2749 CA ALA A 1146−31.782 54.265 32.437 1.00 70.12 C ATOM 2750 C ALA A 1146 −31.211 52.90632.844 1.00 68.68 C ATOM 2751 O ALA A 1146 −30.039 52.815 33.194 1.0070.80 O ATOM 2752 CB ALA A 1146 −32.534 54.894 33.547 1.00 66.71 C ATOM2753 N LYS A 1147 −32.029 51.862 32.786 1.00 69.92 N ATOM 2754 CA LYS A1147 −31.583 50.524 33.161 1.00 72.10 C ATOM 2755 C LYS A 1147 −30.36550.177 32.322 1.00 72.04 C ATOM 2756 O LYS A 1147 −29.317 49.794 32.8811.00 66.93 O ATOM 2757 CB LYS A 1147 −32.712 49.514 32.992 1.00 73.45 CATOM 2758 CG LYS A 1147 −32.536 48.177 33.665 1.00 78.51 C ATOM 2759 CDLYS A 1147 −33.835 47.348 33.477 1.00 82.18 C ATOM 2760 CE LYS A 1147−33.762 45.956 34.103 1.00 93.88 C ATOM 2761 NZ LYS A 1147 −33.81645.967 35.617 1.00 96.78 N ATOM 2762 N ARG A 1148 −30.494 50.323 30.9861.00 73.00 N ATOM 2763 CA ARG A 1148 −29.393 50.016 30.051 1.00 70.89 CATOM 2764 C ARG A 1148 −28.135 50.860 30.239 1.00 74.78 C ATOM 2765 OARG A 1148 −27.034 50.334 30.098 1.00 80.02 O ATOM 2766 CB ARG A 1148−29.832 50.150 28.590 1.00 72.03 C ATOM 2767 CG ARG A 1148 −30.78049.068 28.099 1.00 70.33 C ATOM 2768 CD ARG A 1148 −30.936 49.102 26.5961.00 72.06 C ATOM 2769 NE ARG A 1148 −31.674 50.290 26.158 1.00 71.65 NATOM 2770 CZ ARG A 1148 −33.006 50.391 26.084 1.00 72.14 C ATOM 2771 NH1ARG A 1148 −33.801 49.382 26.416 1.00 78.11 N ATOM 2772 NH2 ARG A 1148−33.568 51.523 25.671 1.00 71.64 N ATOM 2773 N VAL A 1149 −28.270 52.15030.547 1.00 74.24 N ATOM 2774 CA VAL A 1149 −27.072 52.990 30.752 1.0071.90 C ATOM 2775 C VAL A 1149 −26.399 52.632 32.098 1.00 74.21 C ATOM2776 O VAL A 1149 −25.188 52.478 32.164 1.00 77.27 O ATOM 2777 CB VAL A1149 −27.374 54.510 30.676 1.00 78.88 C ATOM 2778 CG1 VAL A 1149 −26.12255.316 31.006 1.00 78.08 C ATOM 2779 CG2 VAL A 1149 −27.939 54.92229.282 1.00 63.48 C ATOM 2780 N ILE A 1150 −27.182 52.498 33.158 1.0070.73 N ATOM 2781 CA ILE A 1150 −26.623 52.164 34.467 1.00 70.91 C ATOM2782 C ILE A 1150 −25.901 50.823 34.437 1.00 69.25 C ATOM 2783 O ILE A1150 −24.744 50.727 34.864 1.00 74.32 O ATOM 2784 CB ILE A 1150 −27.68952.145 35.570 1.00 71.99 C ATOM 2785 CG1 ILE A 1150 −28.264 53.54135.793 1.00 69.99 C ATOM 2786 CG2 ILE A 1150 −27.077 51.645 36.848 1.0070.14 C ATOM 2787 CD1 ILE A 1150 −29.550 53.539 36.637 1.00 76.32 C ATOM2788 N THR A 1151 −26.578 49.793 33.938 1.00 67.61 N ATOM 2789 CA THR A1151 −25.954 48.476 33.707 1.00 67.20 C ATOM 2790 C THR A 1151 −24.64848.608 32.920 1.00 70.88 C ATOM 2791 O THR A 1151 −23.683 47.887 33.1901.00 74.26 O ATOM 2792 CB THR A 1151 −26.876 47.558 32.905 1.00 68.20 CATOM 2793 OG1 THR A 1151 −28.158 47.491 33.541 1.00 72.09 O ATOM 2794CG2 THR A 1151 −26.270 46.158 32.776 1.00 64.27 C ATOM 2795 N THR A 1152−24.619 49.527 31.941 1.00 67.75 N ATOM 2796 CA THR A 1152 −23.39949.788 31.157 1.00 70.23 C ATOM 2797 C THR A 1152 −22.322 50.417 32.0421.00 69.27 C ATOM 2798 O THR A 1152 −21.145 50.068 31.924 1.00 71.67 OATOM 2799 CB THR A 1152 −23.676 50.696 29.941 1.00 66.28 C ATOM 2800 OG1THR A 1152 −24.680 50.076 29.121 1.00 70.58 O ATOM 2801 CG2 THR A 1152−22.396 50.939 29.157 1.00 69.86 C ATOM 2802 N PHE A 1153 −22.719 51.33732.913 1.00 68.71 N ATOM 2803 CA PHE A 1153 −21.791 51.796 33.990 1.0074.06 C ATOM 2804 C PHE A 1153 −21.345 50.649 34.890 1.00 70.59 C ATOM2805 O PHE A 1153 −20.232 50.662 35.389 1.00 76.73 O ATOM 2806 CB PHE A1153 −22.391 52.892 34.872 1.00 71.58 C ATOM 2807 CG PHE A 1153 −22.35954.274 34.258 1.00 75.33 C ATOM 2808 CD1 PHE A 1153 −21.180 54.80733.778 1.00 76.55 C ATOM 2809 CD2 PHE A 1153 −23.508 55.034 34.170 1.0079.26 C ATOM 2810 CE1 PHE A 1153 −21.147 56.059 33.224 1.00 73.15 C ATOM2811 CE2 PHE A 1153 −23.479 56.282 33.622 1.00 72.03 C ATOM 2812 CZ PHEA 1153 −22.296 56.799 33.147 1.00 71.84 C ATOM 2813 N ARG A 1154 −22.19849.655 35.108 1.00 70.31 N ATOM 2814 CA ARG A 1154 −21.801 48.490 35.9401.00 72.56 C ATOM 2815 C ARG A 1154 −20.774 47.559 35.312 1.00 74.07 CATOM 2816 O ARG A 1154 −19.826 47.158 35.994 1.00 75.46 O ATOM 2817 CBARG A 1154 −23.013 47.661 36.354 1.00 71.60 C ATOM 2818 CG ARG A 1154−23.926 48.392 37.315 1.00 78.93 C ATOM 2819 CD ARG A 1154 −24.31347.484 38.485 1.00 80.49 C ATOM 2820 NE ARG A 1154 −25.312 46.492 38.1261.00 81.98 N ATOM 2821 CZ ARG A 1154 −25.657 45.436 38.870 1.00 84.48 CATOM 2822 NH1 ARG A 1154 −25.082 45.186 40.063 1.00 90.99 N ATOM 2823NH2 ARG A 1154 −26.595 44.601 38.421 1.00 88.13 N ATOM 2824 N THR A 1155−20.956 47.219 34.029 1.00 73.12 N ATOM 2825 CA THR A 1155 −20.09546.232 33.346 1.00 74.07 C ATOM 2826 C THR A 1155 −18.969 46.828 32.5011.00 76.10 C ATOM 2827 O THR A 1155 −17.912 46.218 32.361 1.00 76.98 OATOM 2828 CB THR A 1155 −20.940 45.324 32.422 1.00 75.54 C ATOM 2829 OG1THR A 1155 −21.368 46.063 31.251 1.00 76.27 O ATOM 2830 CG2 THR A 1155−22.152 44.777 33.176 1.00 73.31 C ATOM 2831 N GLY A 1156 −19.185 48.00831.936 1.00 75.88 N ATOM 2832 CA GLY A 1156 −18.198 48.591 31.038 1.0076.99 C ATOM 2833 C GLY A 1156 −18.113 47.829 29.703 1.00 78.89 C ATOM2834 O GLY A 1156 −17.054 47.785 29.073 1.00 77.75 O ATOM 2835 N THR A1157 −19.240 47.235 29.297 1.00 79.45 N ATOM 2836 CA THR A 1157 −19.38846.573 28.025 1.00 75.30 C ATOM 2837 C THR A 1157 −20.713 47.007 27.4351.00 76.85 C ATOM 2838 O THR A 1157 −21.568 47.591 28.121 1.00 72.71 OATOM 2839 CB THR A 1157 −19.491 45.069 28.150 1.00 77.35 C ATOM 2840 OG1THR A 1157 −20.771 44.747 28.718 1.00 78.06 O ATOM 2841 CG2 THR A 1157−18.332 44.460 29.016 1.00 73.49 C ATOM 2842 N TRP A 1158 −20.880 46.70626.149 1.00 75.57 N ATOM 2843 CA TRP A 1158 −22.102 47.000 25.412 1.0072.41 C ATOM 2844 C TRP A 1158 −23.213 45.956 25.579 1.00 72.74 C ATOM2845 O TRP A 1158 −24.243 46.070 24.904 1.00 74.46 O ATOM 2846 CB TRP A1158 −21.769 47.101 23.927 1.00 76.07 C ATOM 2847 CG TRP A 1158 −20.84448.147 23.646 1.00 74.03 C ATOM 2848 CD1 TRP A 1158 −19.563 48.00723.274 1.00 71.12 C ATOM 2849 CD2 TRP A 1158 −21.107 49.539 23.717 1.0072.83 C ATOM 2850 NE1 TRP A 1158 −18.986 49.238 23.096 1.00 76.80 N ATOM2851 CE2 TRP A 1158 −19.916 50.200 23.362 1.00 66.00 C ATOM 2852 CE3 TRPA 1158 −22.238 50.297 24.049 1.00 74.60 C ATOM 2853 CZ2 TRP A 1158−19.811 51.582 23.322 1.00 71.92 C ATOM 2854 CZ3 TRP A 1158 −22.13851.673 24.012 1.00 76.31 C ATOM 2855 CH2 TRP A 1158 −20.927 52.30723.649 1.00 74.45 C ATOM 2856 N ASP A 1159 −23.019 44.955 26.460 1.0072.02 N ATOM 2857 CA ASP A 1159 −23.967 43.835 26.664 1.00 71.28 C ATOM2858 C ASP A 1159 −25.450 44.207 26.837 1.00 72.00 C ATOM 2859 O ASP A1159 −26.316 43.422 26.450 1.00 73.98 O ATOM 2860 CB ASP A 1159 −23.52742.966 27.852 1.00 73.56 C ATOM 2861 CG ASP A 1159 −22.254 42.143 27.5701.00 78.31 C ATOM 2862 OD1 ASP A 1159 −21.715 42.178 26.450 1.00 78.77 OATOM 2863 OD2 ASP A 1159 −21.791 41.449 28.499 1.00 92.78 O ATOM 2864 NALA A 1160 −25.746 45.381 27.405 1.00 72.33 N ATOM 2865 CA ALA A 1160−27.154 45.828 27.567 1.00 72.44 C ATOM 2866 C ALA A 1160 −27.815 46.21526.238 1.00 72.65 C ATOM 2867 O ALA A 1160 −29.037 46.285 26.169 1.0075.90 O ATOM 2868 CB ALA A 1160 −27.254 46.998 28.560 1.00 69.43 C ATOM2869 N TYR A 1161 −27.010 46.465 25.200 1.00 73.56 N ATOM 2870 CA TYR A1161 −27.500 46.850 23.870 1.00 70.85 C ATOM 2871 C TYR A 1161 −27.50045.693 22.845 1.00 71.12 C ATOM 2872 O TYR A 1161 −27.779 45.928 21.6841.00 74.87 O ATOM 2873 CB TYR A 1161 −26.712 48.067 23.381 1.00 70.71 CATOM 2874 CG TYR A 1161 −26.977 49.224 24.282 1.00 69.57 C ATOM 2875 CD1TYR A 1161 −28.020 50.090 24.037 1.00 67.53 C ATOM 2876 CD2 TYR A 1161−26.187 49.451 25.400 1.00 71.83 C ATOM 2877 CE1 TYR A 1161 −28.26951.157 24.878 1.00 69.80 C ATOM 2878 CE2 TYR A 1161 −26.432 50.50926.236 1.00 64.36 C ATOM 2879 CZ TYR A 1161 −27.470 51.366 25.980 1.0073.12 C ATOM 2880 OH TYR A 1161 −27.695 52.436 26.850 1.00 77.23 O ATOM2881 N LYS A 263 −27.193 44.468 23.289 1.00 68.62 N ATOM 2882 CA LYS A263 −27.211 43.302 22.437 1.00 68.39 C ATOM 2883 C LYS A 263 −28.61742.992 22.039 1.00 68.68 C ATOM 2884 O LYS A 263 −29.497 43.032 22.8731.00 71.28 O ATOM 2885 CB LYS A 263 −26.716 42.052 23.152 1.00 70.24 CATOM 2886 CG LYS A 263 −25.247 41.957 23.391 1.00 76.63 C ATOM 2887 CDLYS A 263 −24.916 40.567 23.966 1.00 77.14 C ATOM 2888 CE LYS A 263−23.433 40.257 23.926 1.00 86.04 C ATOM 2889 NZ LYS A 263 −23.139 38.78124.083 1.00 88.05 N ATOM 2890 N PHE A 264 −28.823 42.683 20.766 1.0066.31 N ATOM 2891 CA PHE A 264 −30.129 42.267 20.240 1.00 67.14 C ATOM2892 C PHE A 264 −31.247 43.272 20.499 1.00 66.08 C ATOM 2893 O PHE A264 −32.385 42.892 20.754 1.00 66.16 O ATOM 2894 CB PHE A 264 −30.49040.877 20.774 1.00 65.00 C ATOM 2895 CG PHE A 264 −29.455 39.843 20.4611.00 68.77 C ATOM 2896 CD1 PHE A 264 −28.761 39.182 21.465 1.00 69.39 CATOM 2897 CD2 PHE A 264 −29.166 39.530 19.135 1.00 69.77 C ATOM 2898 CE1PHE A 264 −27.800 38.223 21.148 1.00 67.15 C ATOM 2899 CE2 PHE A 264−28.210 38.578 18.825 1.00 68.88 C ATOM 2900 CZ PHE A 264 −27.531 37.92519.832 1.00 67.43 C ATOM 2901 N CYS A 265 −30.891 44.551 20.430 1.0066.76 N ATOM 2902 CA CYS A 265 −31.837 45.636 20.494 1.00 66.33 C ATOM2903 C CYS A 265 −32.192 46.034 19.051 1.00 66.21 C ATOM 2904 O CYS A265 −33.355 46.217 18.742 1.00 67.25 O ATOM 2905 CB CYS A 265 −31.26946.818 21.274 1.00 65.09 C ATOM 2906 SG CYS A 265 −31.066 46.521 23.1101.00 69.25 S ATOM 2907 N LEU A 266 −31.183 46.163 18.186 1.00 63.62 NATOM 2908 CA LEU A 266 −31.385 46.560 16.806 1.00 65.11 C ATOM 2909 CLEU A 266 −32.130 45.487 16.037 1.00 65.91 C ATOM 2910 O LEU A 266−31.815 44.312 16.172 1.00 65.41 O ATOM 2911 CB LEU A 266 −30.044 46.78716.116 1.00 63.47 C ATOM 2912 CG LEU A 266 −29.137 47.928 16.567 1.0066.69 C ATOM 2913 CD1 LEU A 266 −27.791 47.827 15.848 1.00 64.50 C ATOM2914 CD2 LEU A 266 −29.796 49.271 16.325 1.00 66.85 C ATOM 2915 N LYS A267 −33.112 45.898 15.238 1.00 68.75 N ATOM 2916 CA LYS A 267 −33.87044.973 14.350 1.00 71.90 C ATOM 2917 C LYS A 267 −32.973 44.007 13.5661.00 69.09 C ATOM 2918 O LYS A 267 −33.274 42.829 13.463 1.00 72.39 OATOM 2919 CB LYS A 267 −34.768 45.762 13.373 1.00 74.97 C ATOM 2920 CGLYS A 267 −34.008 46.721 12.419 1.00 82.81 C ATOM 2921 CD LYS A 267−34.913 47.656 11.634 1.00 84.17 C ATOM 2922 CE LYS A 267 −34.071 48.65810.816 1.00 88.72 C ATOM 2923 NZ LYS A 267 −34.904 49.777 10.290 1.0088.47 N ATOM 2924 N GLU A 268 −31.877 44.519 13.024 1.00 68.14 N ATOM2925 CA GLU A 268 −30.929 43.706 12.257 1.00 68.69 C ATOM 2926 C GLU A268 −30.232 42.665 13.125 1.00 69.97 C ATOM 2927 O GLU A 268 −30.01041.554 12.679 1.00 71.10 O ATOM 2928 CB GLU A 268 −29.897 44.568 11.4921.00 71.25 C ATOM 2929 CG GLU A 268 −29.063 45.591 12.318 1.00 82.60 CATOM 2930 CD GLU A 268 −29.720 46.975 12.491 1.00 84.41 C ATOM 2931 OE1GLU A 268 −30.934 47.164 12.219 1.00 81.45 O ATOM 2932 OE2 GLU A 268−28.992 47.877 12.911 1.00 83.78 O ATOM 2933 N HIS A 269 −29.891 43.02314.366 1.00 70.32 N ATOM 2934 CA HIS A 269 −29.274 42.065 15.287 1.0068.22 C ATOM 2935 C HIS A 269 −30.290 41.039 15.757 1.00 66.82 C ATOM2936 O HIS A 269 −29.946 39.856 15.882 1.00 65.02 O ATOM 2937 CB HIS A269 −28.612 42.794 16.451 1.00 71.92 C ATOM 2938 CG HIS A 269 −27.39043.554 16.047 1.00 71.60 C ATOM 2939 ND1 HIS A 269 −26.547 44.134 16.9581.00 68.24 N ATOM 2940 CD2 HIS A 269 −26.867 43.824 14.824 1.00 75.13 CATOM 2941 CE1 HIS A 269 −25.557 44.734 16.325 1.00 73.61 C ATOM 2942 NE2HIS A 269 −25.725 44.562 15.027 1.00 78.78 N ATOM 2943 N LYS A 270−31.526 41.477 16.014 1.00 64.48 N ATOM 2944 CA LYS A 270 −32.619 40.53616.296 1.00 67.98 C ATOM 2945 C LYS A 270 −32.761 39.521 15.146 1.0068.82 C ATOM 2946 O LYS A 270 −32.861 38.328 15.394 1.00 66.36 O ATOM2947 CB LYS A 270 −33.948 41.247 16.519 1.00 66.79 C ATOM 2948 CG LYS A270 −33.987 42.127 17.773 1.00 73.23 C ATOM 2949 CD LYS A 270 −35.41242.556 18.088 1.00 71.51 C ATOM 2950 CE LYS A 270 −35.487 43.405 19.3451.00 75.87 C ATOM 2951 NZ LYS A 270 −35.017 42.669 20.545 1.00 77.55 NATOM 2952 N ALA A 271 −32.764 40.009 13.897 1.00 69.99 N ATOM 2953 CAALA A 271 −32.855 39.121 12.713 1.00 71.38 C ATOM 2954 C ALA A 271−31.731 38.111 12.727 1.00 71.12 C ATOM 2955 O ALA A 271 −31.975 36.94612.466 1.00 73.66 O ATOM 2956 CB ALA A 271 −32.855 39.918 11.399 1.0064.88 C ATOM 2957 N LEU A 272 −30.502 38.548 13.034 1.00 72.07 N ATOM2958 CA LEU A 272 −29.359 37.599 13.151 1.00 74.58 C ATOM 2959 C LEU A272 −29.561 36.513 14.240 1.00 75.85 C ATOM 2960 O LEU A 272 −29.13735.367 14.051 1.00 81.93 O ATOM 2961 CB LEU A 272 −28.025 38.324 13.4081.00 73.91 C ATOM 2962 CG LEU A 272 −27.406 39.228 12.329 1.00 78.65 CATOM 2963 CD1 LEU A 272 −26.129 39.864 12.873 1.00 79.09 C ATOM 2964 CD2LEU A 272 −27.118 38.465 11.080 1.00 65.55 C ATOM 2965 N LYS A 273−30.195 36.864 15.358 1.00 76.42 N ATOM 2966 CA LYS A 273 −30.509 35.86616.403 1.00 77.14 C ATOM 2967 C LYS A 273 −31.512 34.845 15.857 1.0077.91 C ATOM 2968 O LYS A 273 −31.346 33.663 16.083 1.00 76.67 O ATOM2969 CB LYS A 273 −31.049 36.529 17.656 1.00 78.11 C ATOM 2970 CG LYS A273 −31.226 35.608 18.867 1.00 80.19 C ATOM 2971 CD LYS A 273 −31.47336.443 20.100 1.00 79.05 C ATOM 2972 CE LYS A 273 −31.752 35.624 21.3291.00 82.30 C ATOM 2973 NZ LYS A 273 −31.953 36.544 22.492 1.00 84.12 NATOM 2974 N THR A 274 −32.541 35.313 15.139 1.00 80.30 N ATOM 2975 CATHR A 274 −33.525 34.414 14.487 1.00 78.23 C ATOM 2976 C THR A 274−32.827 33.369 13.651 1.00 76.51 C ATOM 2977 O THR A 274 −33.082 32.19113.826 1.00 75.03 O ATOM 2978 CB THR A 274 −34.520 35.166 13.564 1.0081.00 C ATOM 2979 OG1 THR A 274 −35.198 36.197 14.293 1.00 83.35 O ATOM2980 CG2 THR A 274 −35.553 34.206 12.963 1.00 79.62 C ATOM 2981 N LEU A275 −31.950 33.800 12.754 1.00 73.92 N ATOM 2982 CA LEU A 275 −31.18332.862 11.925 1.00 74.39 C ATOM 2983 C LEU A 275 −30.330 31.927 12.7791.00 75.45 C ATOM 2984 O LEU A 275 −30.279 30.721 12.524 1.00 76.13 OATOM 2985 CB LEU A 275 −30.301 33.605 10.922 1.00 75.70 C ATOM 2986 CGLEU A 275 −30.975 34.417 9.808 1.00 75.16 C ATOM 2987 CD1 LEU A 275−29.909 35.169 8.981 1.00 67.26 C ATOM 2988 CD2 LEU A 275 −31.846 33.5338.910 1.00 70.95 C ATOM 2989 N GLY A 276 −29.663 32.476 13.797 1.0078.78 N ATOM 2990 CA GLY A 276 −28.890 31.661 14.740 1.00 73.44 C ATOM2991 C GLY A 276 −29.740 30.605 15.422 1.00 73.61 C ATOM 2992 O GLY A276 −29.247 29.515 15.743 1.00 74.08 O ATOM 2993 N ILE A 277 −31.01730.921 15.645 1.00 73.26 N ATOM 2994 CA ILE A 277 −31.955 29.987 16.2861.00 76.95 C ATOM 2995 C ILE A 277 −32.354 28.862 15.334 1.00 75.85 CATOM 2996 O ILE A 277 −32.540 27.720 15.768 1.00 77.29 O ATOM 2997 CBILE A 277 −33.202 30.721 16.866 1.00 74.67 C ATOM 2998 CG1 ILE A 277−32.797 31.558 18.081 1.00 76.85 C ATOM 2999 CG2 ILE A 277 −34.27529.747 17.287 1.00 72.00 C ATOM 3000 CD1 ILE A 277 −33.970 32.239 18.7881.00 76.44 C ATOM 3001 N ILE A 278 −32.481 29.188 14.048 1.00 79.83 NATOM 3002 CA ILE A 278 −32.787 28.208 13.010 1.00 77.27 C ATOM 3003 CILE A 278 −31.630 27.225 12.931 1.00 79.77 C ATOM 3004 O ILE A 278−31.833 26.004 12.877 1.00 80.80 O ATOM 3005 CB ILE A 278 −33.004 28.89211.637 1.00 77.96 C ATOM 3006 CG1 ILE A 278 −34.244 29.798 11.651 1.0079.43 C ATOM 3007 CG2 ILE A 278 −33.174 27.888 10.557 1.00 78.57 C ATOM3008 CD1 ILE A 278 −35.544 29.087 11.775 1.00 83.47 C ATOM 3009 N MET A279 −30.412 27.761 12.933 1.00 79.21 N ATOM 3010 CA MET A 279 −29.20626.942 12.826 1.00 77.35 C ATOM 3011 C MET A 279 −29.019 26.044 14.0181.00 75.85 C ATOM 3012 O MET A 279 −28.798 24.844 13.852 1.00 73.53 OATOM 3013 CB MET A 279 −27.982 27.830 12.632 1.00 76.42 C ATOM 3014 CGMET A 279 −27.992 28.535 11.284 1.00 79.03 C ATOM 3015 SD MET A 279−26.852 29.897 11.156 1.00 80.66 S ATOM 3016 CE MET A 279 −27.061 30.3049.407 1.00 79.86 C ATOM 3017 N GLY A 280 −29.112 26.625 15.214 1.0078.82 N ATOM 3018 CA GLY A 280 −28.937 25.880 16.463 1.00 76.24 C ATOM3019 C GLY A 280 −29.970 24.785 16.660 1.00 79.31 C ATOM 3020 O GLY A280 −29.640 23.694 17.120 1.00 80.01 O ATOM 3021 N THR A 281 −31.21925.084 16.309 1.00 80.59 N ATOM 3022 CA THR A 281 −32.309 24.123 16.4081.00 78.83 C ATOM 3023 C THR A 281 −32.057 22.935 15.492 1.00 77.28 CATOM 3024 O THR A 281 −32.190 21.801 15.923 1.00 76.07 O ATOM 3025 CBTHR A 281 −33.657 24.780 16.070 1.00 81.71 C ATOM 3026 OG1 THR A 281−33.964 25.771 17.065 1.00 79.40 O ATOM 3027 CG2 THR A 281 −34.76923.760 16.038 1.00 82.36 C ATOM 3028 N PHE A 282 −31.694 23.205 14.2371.00 79.67 N ATOM 3029 CA PHE A 282 −31.347 22.147 13.279 1.00 77.00 CATOM 3030 C PHE A 282 −30.221 21.282 13.817 1.00 76.25 C ATOM 3031 O PHEA 282 −30.266 20.069 13.697 1.00 73.80 O ATOM 3032 CB PHE A 282 −30.91722.731 11.930 1.00 77.95 C ATOM 3033 CG PHE A 282 −30.691 21.679 10.8531.00 76.73 C ATOM 3034 CD1 PHE A 282 −31.723 21.314 9.995 1.00 75.49 CATOM 3035 CD2 PHE A 282 −29.460 21.064 10.706 1.00 76.37 C ATOM 3036 CE1PHE A 282 −31.526 20.343 8.997 1.00 76.36 C ATOM 3037 CE2 PHE A 282−29.254 20.097 9.720 1.00 84.43 C ATOM 3038 CZ PHE A 282 −30.297 19.7378.857 1.00 76.69 C ATOM 3039 N THR A 283 −29.214 21.923 14.406 1.0077.11 N ATOM 3040 CA THR A 283 −28.058 21.218 14.945 1.00 76.40 C ATOM3041 C THR A 283 −28.460 20.311 16.078 1.00 76.62 C ATOM 3042 O THR A283 −28.105 19.152 16.073 1.00 76.69 O ATOM 3043 CB THR A 283 −26.98122.183 15.448 1.00 76.06 C ATOM 3044 OG1 THR A 283 −26.544 23.023 14.3671.00 80.64 O ATOM 3045 CG2 THR A 283 −25.784 21.412 16.018 1.00 75.59 CATOM 3046 N LEU A 284 −29.203 20.844 17.041 1.00 78.59 N ATOM 3047 CALEU A 284 −29.636 20.065 18.204 1.00 77.27 C ATOM 3048 C LEU A 284−30.578 18.920 17.845 1.00 77.57 C ATOM 3049 O LEU A 284 −30.560 17.88618.500 1.00 76.25 O ATOM 3050 CB LEU A 284 −30.300 20.986 19.236 1.0080.83 C ATOM 3051 CG LEU A 284 −29.359 22.013 19.916 1.00 89.13 C ATOM3052 CD1 LEU A 284 −30.162 23.029 20.724 1.00 93.20 C ATOM 3053 CD2 LEUA 284 −28.290 21.337 20.801 1.00 91.45 C ATOM 3054 N CYS A 285 −31.39519.108 16.806 1.00 77.86 N ATOM 3055 CA CYS A 285 −32.341 18.073 16.3671.00 76.99 C ATOM 3056 C CYS A 285 −31.677 16.920 15.620 1.00 75.92 CATOM 3057 O CYS A 285 −32.095 15.782 15.783 1.00 75.32 O ATOM 3058 CBCYS A 285 −33.462 18.678 15.513 1.00 76.74 C ATOM 3059 SG CYS A 285−34.727 19.541 16.511 1.00 86.35 S ATOM 3060 N TRP A 286 −30.657 17.21914.810 1.00 76.29 N ATOM 3061 CA TRP A 286 −29.938 16.192 14.020 1.0075.01 C ATOM 3062 C TRP A 286 −28.635 15.671 14.635 1.00 75.15 C ATOM3063 O TRP A 286 −28.250 14.544 14.330 1.00 75.31 O ATOM 3064 CB TRP A286 −29.641 16.703 12.599 1.00 72.75 C ATOM 3065 CG TRP A 286 −30.85316.738 11.766 1.00 70.37 C ATOM 3066 CD1 TRP A 286 −31.543 17.832 11.3811.00 69.67 C ATOM 3067 CD2 TRP A 286 −31.533 15.613 11.212 1.00 67.81 CATOM 3068 NE1 TRP A 286 −32.621 17.469 10.614 1.00 76.08 N ATOM 3069 CE2TRP A 286 −32.636 16.107 10.496 1.00 67.36 C ATOM 3070 CE3 TRP A 286−31.314 14.228 11.254 1.00 69.38 C ATOM 3071 CZ2 TRP A 286 −33.52315.274 9.822 1.00 73.52 C ATOM 3072 CZ3 TRP A 286 −32.202 13.391 10.5811.00 71.27 C ATOM 3073 CH2 TRP A 286 −33.291 13.920 9.875 1.00 72.45 CATOM 3074 N LEU A 287 −27.958 16.454 15.474 1.00 71.19 N ATOM 3075 CALEU A 287 −26.645 16.020 16.001 1.00 72.17 C ATOM 3076 C LEU A 287−26.669 14.651 16.667 1.00 70.61 C ATOM 3077 O LEU A 287 −25.822 13.83416.347 1.00 73.46 O ATOM 3078 CB LEU A 287 −26.019 17.049 16.953 1.0074.35 C ATOM 3079 CG LEU A 287 −24.542 16.884 17.277 1.00 76.88 C ATOM3080 CD1 LEU A 287 −23.682 16.964 16.013 1.00 78.66 C ATOM 3081 CD2 LEUA 287 −24.130 17.956 18.279 1.00 79.06 C ATOM 3082 N PRO A 288 −27.62714.395 17.583 1.00 69.58 N ATOM 3083 CA PRO A 288 −27.682 13.074 18.2271.00 68.99 C ATOM 3084 C PRO A 288 −27.767 11.903 17.248 1.00 68.90 CATOM 3085 O PRO A 288 −27.052 10.906 17.406 1.00 67.39 O ATOM 3086 CBPRO A 288 −28.979 13.140 19.057 1.00 68.33 C ATOM 3087 CG PRO A 288−29.207 14.546 19.305 1.00 66.28 C ATOM 3088 CD PRO A 288 −28.701 15.26918.091 1.00 70.69 C ATOM 3089 N PHE A 289 −28.641 12.042 16.255 1.0067.58 N ATOM 3090 CA PHE A 289 −28.807 11.038 15.217 1.00 67.91 C ATOM3091 C PHE A 289 −27.479 10.746 14.519 1.00 69.22 C ATOM 3092 O PHE A289 −27.072 9.576 14.412 1.00 68.30 O ATOM 3093 CB PHE A 289 −29.85311.505 14.189 1.00 69.82 C ATOM 3094 CG PHE A 289 −30.014 10.578 13.0471.00 67.38 C ATOM 3095 CD1 PHE A 289 −30.850 9.475 13.157 1.00 64.32 CATOM 3096 CD2 PHE A 289 −29.335 10.795 11.859 1.00 71.12 C ATOM 3097 CE1PHE A 289 −31.008 8.610 12.114 1.00 70.14 C ATOM 3098 CE2 PHE A 289−29.489 9.926 10.798 1.00 76.34 C ATOM 3099 CZ PHE A 289 −30.327 8.82910.923 1.00 75.79 C ATOM 3100 N PHE A 290 −26.818 11.811 14.052 1.0069.03 N ATOM 3101 CA PHE A 290 −25.522 11.686 13.347 1.00 70.29 C ATOM3102 C PHE A 290 −24.346 11.254 14.222 1.00 70.41 C ATOM 3103 O PHE A290 −23.386 10.677 13.697 1.00 72.74 O ATOM 3104 CB PHE A 290 −25.18012.971 12.585 1.00 73.06 C ATOM 3105 CG PHE A 290 −25.942 13.108 11.2951.00 73.80 C ATOM 3106 CD1 PHE A 290 −27.103 13.864 11.226 1.00 70.64 CATOM 3107 CD2 PHE A 290 −25.494 12.467 10.149 1.00 76.58 C ATOM 3108 CE1PHE A 290 −27.800 13.990 10.058 1.00 79.74 C ATOM 3109 CE2 PHE A 290−26.191 12.586 8.960 1.00 75.30 C ATOM 3110 CZ PHE A 290 −27.349 13.3528.914 1.00 77.36 C ATOM 3111 N ILE A 291 −24.404 11.522 15.528 1.0069.93 N ATOM 3112 CA ILE A 291 −23.391 11.003 16.454 1.00 71.11 C ATOM3113 C ILE A 291 −23.517 9.476 16.516 1.00 72.55 C ATOM 3114 O ILE A 291−22.507 8.765 16.534 1.00 76.20 O ATOM 3115 CB ILE A 291 −23.511 11.62117.873 1.00 71.52 C ATOM 3116 CG1 ILE A 291 −23.073 13.081 17.853 1.0073.15 C ATOM 3117 CG2 ILE A 291 −22.642 10.888 18.894 1.00 65.02 C ATOM3118 CD1 ILE A 291 −23.309 13.807 19.140 1.00 72.16 C ATOM 3119 N VAL A292 −24.757 8.984 16.545 1.00 74.02 N ATOM 3120 CA VAL A 292 −25.0237.539 16.619 1.00 74.95 C ATOM 3121 C VAL A 292 −24.586 6.782 15.3401.00 73.29 C ATOM 3122 O VAL A 292 −24.192 5.619 15.426 1.00 74.16 OATOM 3123 CB VAL A 292 −26.505 7.253 16.998 1.00 74.89 C ATOM 3124 CG1VAL A 292 −26.814 5.775 16.915 1.00 73.02 C ATOM 3125 CG2 VAL A 292−26.781 7.774 18.427 1.00 73.75 C ATOM 3126 N ASN A 293 −24.651 7.43114.176 1.00 73.79 N ATOM 3127 CA ASN A 293 −24.112 6.835 12.936 1.0072.79 C ATOM 3128 C ASN A 293 −22.641 6.495 13.085 1.00 71.66 C ATOM3129 O ASN A 293 −22.206 5.409 12.709 1.00 72.11 O ATOM 3130 CB ASN A293 −24.250 7.782 11.745 1.00 72.08 C ATOM 3131 CG ASN A 293 −25.6737.961 11.287 1.00 76.70 C ATOM 3132 OD1 ASN A 293 −26.584 7.306 11.7771.00 77.56 O ATOM 3133 ND2 ASN A 293 −25.871 8.863 10.327 1.00 72.62 NATOM 3134 N ILE A 294 −21.887 7.435 13.639 1.00 70.24 N ATOM 3135 CA ILEA 294 −20.451 7.272 13.833 1.00 71.74 C ATOM 3136 C ILE A 294 −20.1566.296 14.963 1.00 71.03 C ATOM 3137 O ILE A 294 −19.288 5.437 14.8071.00 69.26 O ATOM 3138 CB ILE A 294 −19.784 8.608 14.115 1.00 71.93 CATOM 3139 CG1 ILE A 294 −19.878 9.502 12.885 1.00 71.27 C ATOM 3140 CG2ILE A 294 −18.313 8.421 14.498 1.00 72.08 C ATOM 3141 CD1 ILE A 294−19.391 10.862 13.168 1.00 79.13 C ATOM 3142 N VAL A 295 −20.872 6.42616.088 1.00 71.55 N ATOM 3143 CA VAL A 295 −20.735 5.484 17.234 1.0070.36 C ATOM 3144 C VAL A 295 −20.932 4.029 16.782 1.00 69.42 C ATOM3145 O VAL A 295 −20.224 3.142 17.243 1.00 66.44 O ATOM 3146 CB VAL A295 −21.720 5.835 18.396 1.00 70.22 C ATOM 3147 CG1 VAL A 295 −21.8034.721 19.419 1.00 68.33 C ATOM 3148 CG2 VAL A 295 −21.304 7.125 19.0651.00 71.01 C ATOM 3149 N HIS A 296 −21.890 3.805 15.878 1.00 69.19 NATOM 3150 CA HIS A 296 −22.128 2.462 15.311 1.00 71.67 C ATOM 3151 C HISA 296 −21.079 1.977 14.297 1.00 72.30 C ATOM 3152 O HIS A 296 −21.0220.785 13.996 1.00 72.47 O ATOM 3153 CB HIS A 296 −23.545 2.349 14.7371.00 72.70 C ATOM 3154 CG HIS A 296 −24.591 2.269 15.795 1.00 77.53 CATOM 3155 ND1 HIS A 296 −24.369 1.632 16.991 1.00 78.84 N ATOM 3156 CD2HIS A 296 −25.859 2.736 15.848 1.00 85.05 C ATOM 3157 CE1 HIS A 296−25.448 1.710 17.733 1.00 78.27 C ATOM 3158 NE2 HIS A 296 −26.370 2.37317.069 1.00 81.35 N ATOM 3159 N VAL A 297 −20.267 2.893 13.781 1.0073.43 N ATOM 3160 CA VAL A 297 −19.118 2.535 12.948 1.00 73.61 C ATOM3161 C VAL A 297 −17.993 2.051 13.878 1.00 72.62 C ATOM 3162 O VAL A 297−17.220 1.165 13.505 1.00 73.05 O ATOM 3163 CB VAL A 297 −18.653 3.71312.050 1.00 73.85 C ATOM 3164 CG1 VAL A 297 −17.424 3.338 11.238 1.0072.56 C ATOM 3165 CG2 VAL A 297 −19.786 4.139 11.127 1.00 72.80 C ATOM3166 N ILE A 298 −17.912 2.636 15.082 1.00 72.35 N ATOM 3167 CA ILE A298 −16.930 2.236 16.097 1.00 73.03 C ATOM 3168 C ILE A 298 −17.3510.886 16.688 1.00 73.53 C ATOM 3169 O ILE A 298 −16.612 −0.099 16.5971.00 74.08 O ATOM 3170 CB ILE A 298 −16.780 3.288 17.246 1.00 73.65 CATOM 3171 CG1 ILE A 298 −16.402 4.687 16.716 1.00 76.60 C ATOM 3172 CG2ILE A 298 −15.757 2.827 18.255 1.00 73.25 C ATOM 3173 CD1 ILE A 298−15.059 4.764 16.007 1.00 80.21 C ATOM 3174 N GLN A 299 −18.542 0.86417.290 1.00 73.48 N ATOM 3175 CA GLN A 299 −19.132 −0.345 17.862 1.0074.55 C ATOM 3176 C GLN A 299 −20.619 −0.354 17.524 1.00 75.17 C ATOM3177 O GLN A 299 −21.368 0.466 18.040 1.00 77.55 O ATOM 3178 CB GLN A299 −18.917 −0.401 19.378 1.00 75.38 C ATOM 3179 CG GLN A 299 −19.532−1.630 20.086 1.00 76.95 C ATOM 3180 CD GLN A 299 −18.993 −2.963 19.5631.00 80.36 C ATOM 3181 OE1 GLN A 299 −17.801 −3.255 19.675 1.00 81.59 OATOM 3182 NE2 GLN A 299 −19.883 −3.778 18.992 1.00 79.18 N ATOM 3183 NASP A 300 −21.035 −1.282 16.662 1.00 75.16 N ATOM 3184 CA ASP A 300−22.420 −1.357 16.206 1.00 76.26 C ATOM 3185 C ASP A 300 −23.306 −1.99417.284 1.00 76.70 C ATOM 3186 O ASP A 300 −22.818 −2.756 18.121 1.0075.98 O ATOM 3187 CB ASP A 300 −22.492 −2.145 14.883 1.00 76.84 C ATOM3188 CG ASP A 300 −23.781 −1.873 14.069 1.00 78.37 C ATOM 3189 OD1 ASP A300 −24.504 −0.874 14.303 1.00 80.03 O ATOM 3190 OD2 ASP A 300 −24.068−2.688 13.168 1.00 83.02 O ATOM 3191 N ASN A 301 −24.601 −1.661 17.2481.00 76.86 N ATOM 3192 CA ASN A 301 −25.633 −2.196 18.177 1.00 76.17 CATOM 3193 C ASN A 301 −25.536 −1.740 19.656 1.00 75.65 C ATOM 3194 O ASNA 301 −26.220 −2.290 20.520 1.00 76.59 O ATOM 3195 CB ASN A 301 −25.700−3.741 18.089 1.00 75.63 C ATOM 3196 CG ASN A 301 −26.022 −4.235 16.6901.00 74.71 C ATOM 3197 OD1 ASN A 301 −26.917 −3.714 16.030 1.00 64.90 OATOM 3198 ND2 ASN A 301 −25.291 −5.246 16.233 1.00 75.78 N ATOM 3199 NLEU A 302 −24.700 −0.743 19.938 1.00 76.35 N ATOM 3200 CA LEU A 302−24.547 −0.197 21.298 1.00 78.80 C ATOM 3201 C LEU A 302 −25.815 0.53421.817 1.00 79.26 C ATOM 3202 O LEU A 302 −26.074 0.566 23.018 1.0080.59 O ATOM 3203 CB LEU A 302 −23.350 0.764 21.335 1.00 79.16 C ATOM3204 CG LEU A 302 −22.832 1.213 22.703 1.00 79.37 C ATOM 3205 CD1 LEU A302 −22.199 0.039 23.438 1.00 77.36 C ATOM 3206 CD2 LEU A 302 −21.8392.351 22.545 1.00 79.97 C ATOM 3207 N ILE A 303 −26.572 1.105 20.8881.00 79.64 N ATOM 3208 CA ILE A 303 −27.765 1.886 21.120 1.00 80.47 CATOM 3209 C ILE A 303 −28.881 1.166 20.393 1.00 80.12 C ATOM 3210 O ILEA 303 −28.747 0.854 19.208 1.00 80.56 O ATOM 3211 CB ILE A 303 −27.6233.313 20.573 1.00 79.71 C ATOM 3212 CG1 ILE A 303 −26.513 4.056 21.3261.00 80.55 C ATOM 3213 CG2 ILE A 303 −28.930 4.068 20.708 1.00 78.77 CATOM 3214 CD1 ILE A 303 −26.243 5.427 20.826 1.00 83.25 C ATOM 3215 NARG A 304 −29.973 0.910 21.111 1.00 81.10 N ATOM 3216 CA ARG A 304−31.132 0.177 20.596 1.00 83.66 C ATOM 3217 C ARG A 304 −31.686 0.77619.310 1.00 82.48 C ATOM 3218 O ARG A 304 −31.542 1.977 19.070 1.0083.57 O ATOM 3219 CB ARG A 304 −32.265 0.192 21.608 1.00 84.33 C ATOM3220 CG ARG A 304 −31.969 −0.426 22.966 1.00 91.17 C ATOM 3221 CD ARG A304 −33.142 −0.227 23.870 1.00 96.90 C ATOM 3222 NE ARG A 304 −34.321−0.914 23.347 1.00 107.82 N ATOM 3223 CZ ARG A 304 −35.556 −0.820 23.8451.00 114.88 C ATOM 3224 NH1 ARG A 304 −35.826 −0.056 24.909 1.00 119.98N ATOM 3225 NH2 ARG A 304 −36.539 −1.505 23.268 1.00 117.19 N ATOM 3226N LYS A 305 −32.317 −0.069 18.496 1.00 80.96 N ATOM 3227 CA LYS A 305−32.950 0.369 17.249 1.00 82.83 C ATOM 3228 C LYS A 305 −34.024 1.43217.501 1.00 82.19 C ATOM 3229 O LYS A 305 −34.122 2.399 16.751 1.0083.88 O ATOM 3230 CB LYS A 305 −33.573 −0.819 16.514 1.00 82.92 C ATOM3231 CG LYS A 305 −34.091 −0.499 15.109 1.00 86.35 C ATOM 3232 CD LYS A305 −34.754 −1.709 14.472 1.00 87.13 C ATOM 3233 CE LYS A 305 −35.417−1.329 13.154 1.00 88.05 C ATOM 3234 NZ LYS A 305 −36.147 −2.481 12.5521.00 91.16 N ATOM 3235 N GLU A 306 −34.813 1.237 18.553 1.00 79.77 NATOM 3236 CA GLU A 306 −35.888 2.166 18.928 1.00 78.91 C ATOM 3237 C GLUA 306 −35.388 3.534 19.354 1.00 75.42 C ATOM 3238 O GLU A 306 −36.0834.526 19.156 1.00 73.51 O ATOM 3239 CB GLU A 306 −36.707 1.586 20.0621.00 80.27 C ATOM 3240 CG GLU A 306 −37.464 0.323 19.682 1.00 88.15 CATOM 3241 CD GLU A 306 −38.035 −0.379 20.876 1.00 92.42 C ATOM 3242 OE1GLU A 306 −38.087 0.245 21.964 1.00 103.55 O ATOM 3243 OE2 GLU A 306−38.436 −1.560 20.740 1.00 104.10 O ATOM 3244 N VAL A 307 −34.191 3.58619.938 1.00 74.31 N ATOM 3245 CA VAL A 307 −33.579 4.864 20.314 1.0073.09 C ATOM 3246 C VAL A 307 −33.126 5.563 19.040 1.00 72.26 C ATOM3247 O VAL A 307 −33.415 6.740 18.854 1.00 74.87 O ATOM 3248 CB VAL A307 −32.398 4.703 21.330 1.00 71.64 C ATOM 3249 CG1 VAL A 307 −31.5975.986 21.441 1.00 63.03 C ATOM 3250 CG2 VAL A 307 −32.925 4.287 22.7031.00 68.24 C ATOM 3251 N TYR A 308 −32.423 4.833 18.177 1.00 71.72 NATOM 3252 CA TYR A 308 −31.955 5.366 16.892 1.00 72.58 C ATOM 3253 C TYRA 308 −33.106 5.892 16.025 1.00 72.20 C ATOM 3254 O TYR A 308 −32.9816.951 15.414 1.00 72.72 O ATOM 3255 CB TYR A 308 −31.184 4.294 16.1351.00 75.16 C ATOM 3256 CG TYR A 308 −30.534 4.770 14.855 1.00 75.61 CATOM 3257 CD1 TYR A 308 −30.984 4.338 13.610 1.00 83.25 C ATOM 3258 CD2TYR A 308 −29.470 5.650 14.894 1.00 77.87 C ATOM 3259 CE1 TYR A 308−30.378 4.780 12.435 1.00 82.87 C ATOM 3260 CE2 TYR A 308 −28.859 6.09813.747 1.00 74.78 C ATOM 3261 CZ TYR A 308 −29.313 5.663 12.513 1.0080.83 C ATOM 3262 OH TYR A 308 −28.716 6.100 11.357 1.00 79.13 O ATOM3263 N ILE A 309 −34.214 5.152 15.979 1.00 70.18 N ATOM 3264 CA ILE A309 −35.412 5.593 15.251 1.00 71.74 C ATOM 3265 C ILE A 309 −35.9596.873 15.863 1.00 72.46 C ATOM 3266 O ILE A 309 −36.272 7.798 15.1391.00 73.87 O ATOM 3267 CB ILE A 309 −36.519 4.520 15.243 1.00 70.27 CATOM 3268 CG1 ILE A 309 −36.141 3.375 14.316 1.00 69.96 C ATOM 3269 CG2ILE A 309 −37.839 5.100 14.781 1.00 68.79 C ATOM 3270 CD1 ILE A 309−36.987 2.145 14.516 1.00 72.32 C ATOM 3271 N LEU A 310 −36.067 6.91717.195 1.00 74.30 N ATOM 3272 CA LEU A 310 −36.561 8.109 17.895 1.0073.71 C ATOM 3273 C LEU A 310 −35.682 9.324 17.619 1.00 75.46 C ATOM3274 O LEU A 310 −36.208 10.416 17.414 1.00 76.09 O ATOM 3275 CB LEU A310 −36.677 7.873 19.405 1.00 76.16 C ATOM 3276 CG LEU A 310 −37.1039.087 20.269 1.00 79.17 C ATOM 3277 CD1 LEU A 310 −38.353 9.787 19.6991.00 80.03 C ATOM 3278 CD2 LEU A 310 −37.344 8.666 21.710 1.00 74.92 CATOM 3279 N LEU A 311 −34.358 9.142 17.612 1.00 74.63 N ATOM 3280 CA LEUA 311 −33.436 10.249 17.271 1.00 73.90 C ATOM 3281 C LEU A 311 −33.61110.744 15.829 1.00 73.58 C ATOM 3282 O LEU A 311 −33.346 11.904 15.5461.00 75.24 O ATOM 3283 CB LEU A 311 −31.976 9.857 17.497 1.00 71.95 CATOM 3284 CG LEU A 311 −31.583 9.468 18.926 1.00 74.42 C ATOM 3285 CD1LEU A 311 −30.094 9.202 18.969 1.00 78.05 C ATOM 3286 CD2 LEU A 311−31.960 10.525 19.951 1.00 79.68 C ATOM 3287 N ASN A 312 −34.053 9.86214.932 1.00 73.28 N ATOM 3288 CA ASN A 312 −34.337 10.228 13.551 1.0074.59 C ATOM 3289 C ASN A 312 −35.602 11.076 13.491 1.00 74.96 C ATOM3290 O ASN A 312 −35.654 12.057 12.741 1.00 76.71 O ATOM 3291 CB ASN A312 −34.490 8.970 12.679 1.00 73.89 C ATOM 3292 CG ASN A 312 −34.2769.231 11.170 1.00 75.40 C ATOM 3293 OD1 ASN A 312 −33.933 8.302 10.4391.00 75.69 O ATOM 3294 ND2 ASN A 312 −34.471 10.471 10.709 1.00 73.01 NATOM 3295 N TRP A 313 −36.614 10.702 14.275 1.00 75.65 N ATOM 3296 CATRP A 313 −37.880 11.461 14.327 1.00 73.90 C ATOM 3297 C TRP A 313−37.748 12.844 14.967 1.00 74.02 C ATOM 3298 O TRP A 313 −38.556 13.72614.676 1.00 74.92 O ATOM 3299 CB TRP A 313 −38.988 10.634 14.986 1.0075.64 C ATOM 3300 CG TRP A 313 −39.562 9.768 13.984 1.00 77.34 C ATOM3301 CD1 TRP A 313 −39.070 8.583 13.548 1.00 77.95 C ATOM 3302 CD2 TRP A313 −40.757 10.004 13.254 1.00 77.66 C ATOM 3303 NE1 TRP A 313 −39.8858.056 12.589 1.00 77.47 N ATOM 3304 CE2 TRP A 313 −40.935 8.910 12.3841.00 79.44 C ATOM 3305 CE3 TRP A 313 −41.701 11.037 13.247 1.00 76.72 CATOM 3306 CZ2 TRP A 313 −42.025 8.813 11.509 1.00 79.40 C ATOM 3307 CZ3TRP A 313 −42.790 10.944 12.374 1.00 78.33 C ATOM 3308 CH2 TRP A 313−42.940 9.835 11.518 1.00 77.48 C ATOM 3309 N ILE A 314 −36.742 13.02415.822 1.00 71.91 N ATOM 3310 CA ILE A 314 −36.411 14.336 16.368 1.0071.31 C ATOM 3311 C ILE A 314 −35.847 15.185 15.213 1.00 71.78 C ATOM3312 O ILE A 314 −36.108 16.396 15.114 1.00 72.42 O ATOM 3313 CB ILE A314 −35.406 14.239 17.546 1.00 69.93 C ATOM 3314 CG1 ILE A 314 −36.04013.519 18.735 1.00 69.22 C ATOM 3315 CG2 ILE A 314 −34.975 15.611 18.0021.00 66.77 C ATOM 3316 CD1 ILE A 314 −35.054 13.195 19.841 1.00 70.69 CATOM 3317 N GLY A 315 −35.072 14.536 14.350 1.00 71.45 N ATOM 3318 CAGLY A 315 −34.603 15.150 13.118 1.00 73.83 C ATOM 3319 C GLY A 315−35.780 15.520 12.237 1.00 73.35 C ATOM 3320 O GLY A 315 −35.913 16.67311.866 1.00 71.64 O ATOM 3321 N TYR A 316 −36.634 14.545 11.909 1.0074.36 N ATOM 3322 CA TYR A 316 −37.825 14.800 11.050 1.00 74.23 C ATOM3323 C TYR A 316 −38.668 15.970 11.526 1.00 75.48 C ATOM 3324 O TYR A316 −38.949 16.889 10.754 1.00 76.19 O ATOM 3325 CB TYR A 316 −38.74613.570 10.960 1.00 74.65 C ATOM 3326 CG TYR A 316 −38.256 12.359 10.1391.00 77.26 C ATOM 3327 CD1 TYR A 316 −38.869 11.121 10.288 1.00 77.77 CATOM 3328 CD2 TYR A 316 −37.200 12.448 9.231 1.00 75.43 C ATOM 3329 CE1TYR A 316 −38.459 10.025 9.571 1.00 71.53 C ATOM 3330 CE2 TYR A 316−36.787 11.358 8.514 1.00 78.76 C ATOM 3331 CZ TYR A 316 −37.420 10.1488.688 1.00 77.38 C ATOM 3332 OH TYR A 316 −37.015 9.061 7.979 1.00 77.21O ATOM 3333 N VAL A 317 −39.057 15.910 12.801 1.00 74.20 N ATOM 3334 CAVAL A 317 −39.872 16.934 13.466 1.00 72.51 C ATOM 3335 C VAL A 317−39.316 18.355 13.330 1.00 72.64 C ATOM 3336 O VAL A 317 −40.086 19.31113.253 1.00 72.17 O ATOM 3337 CB VAL A 317 −40.088 16.560 14.984 1.0072.14 C ATOM 3338 CG1 VAL A 317 −40.336 17.760 15.804 1.00 74.78 C ATOM3339 CG2 VAL A 317 −41.253 15.603 15.111 1.00 69.59 C ATOM 3340 N ASN A318 −37.988 18.491 13.296 1.00 74.61 N ATOM 3341 CA ASN A 318 −37.34219.793 13.078 1.00 75.44 C ATOM 3342 C ASN A 318 −37.882 20.548 11.8571.00 75.25 C ATOM 3343 O ASN A 318 −37.912 21.779 11.844 1.00 72.63 OATOM 3344 CB ASN A 318 −35.843 19.645 12.910 1.00 74.36 C ATOM 3345 CGASN A 318 −35.171 20.958 12.635 1.00 79.05 C ATOM 3346 OD1 ASN A 318−35.023 21.806 13.522 1.00 80.80 O ATOM 3347 ND2 ASN A 318 −34.76121.142 11.396 1.00 83.57 N ATOM 3348 N SER A 319 −38.308 19.798 10.8361.00 75.32 N ATOM 3349 CA SER A 319 −38.951 20.365 9.640 1.00 76.04 CATOM 3350 C SER A 319 −40.218 21.194 9.925 1.00 76.24 C ATOM 3351 O SERA 319 −40.670 21.930 9.046 1.00 77.32 O ATOM 3352 CB SER A 319 −39.28519.245 8.635 1.00 74.82 C ATOM 3353 OG SER A 319 −38.120 18.509 8.2811.00 77.99 O ATOM 3354 N GLY A 320 −40.782 21.078 11.133 1.00 75.25 NATOM 3355 CA GLY A 320 −41.934 21.857 11.544 1.00 74.43 C ATOM 3356 CGLY A 320 −41.642 23.110 12.350 1.00 76.72 C ATOM 3357 O GLY A 320−42.562 23.884 12.581 1.00 78.81 O ATOM 3358 N PHE A 321 −40.391 23.32812.777 1.00 75.79 N ATOM 3359 CA PHE A 321 −40.076 24.440 13.706 1.0077.01 C ATOM 3360 C PHE A 321 −39.761 25.780 13.112 1.00 77.35 C ATOM3361 O PHE A 321 −40.178 26.799 13.680 1.00 75.78 O ATOM 3362 CB PHE A321 −38.912 24.051 14.634 1.00 78.53 C ATOM 3363 CG PHE A 321 −39.19222.851 15.497 1.00 78.83 C ATOM 3364 CD1 PHE A 321 −38.154 22.005 15.8571.00 82.01 C ATOM 3365 CD2 PHE A 321 −40.490 22.551 15.953 1.00 81.40 CATOM 3366 CE1 PHE A 321 −38.383 20.891 16.650 1.00 81.76 C ATOM 3367 CE2PHE A 321 −40.722 21.446 16.740 1.00 83.47 C ATOM 3368 CZ PHE A 321−39.667 20.614 17.091 1.00 85.30 C ATOM 3369 N ASN A 322 −39.040 25.80711.996 1.00 77.86 N ATOM 3370 CA ASN A 322 −38.637 27.082 11.381 1.0078.02 C ATOM 3371 C ASN A 322 −39.785 28.061 11.142 1.00 78.43 C ATOM3372 O ASN A 322 −39.620 29.237 11.440 1.00 77.20 O ATOM 3373 CB ASN A322 −37.866 26.856 10.087 1.00 79.19 C ATOM 3374 CG ASN A 322 −36.53126.208 10.315 1.00 81.79 C ATOM 3375 OD1 ASN A 322 −36.158 25.901 11.4551.00 74.61 O ATOM 3376 ND2 ASN A 322 −35.791 25.989 9.229 1.00 85.04 NATOM 3377 N PRO A 323 −40.941 27.582 10.609 1.00 78.55 N ATOM 3378 CAPRO A 323 −42.088 28.479 10.469 1.00 79.23 C ATOM 3379 C PRO A 323−42.469 29.179 11.779 1.00 80.55 C ATOM 3380 O PRO A 323 −42.795 30.36711.751 1.00 83.61 O ATOM 3381 CB PRO A 323 −43.200 27.542 9.997 1.0080.04 C ATOM 3382 CG PRO A 323 −42.474 26.473 9.266 1.00 79.60 C ATOM3383 CD PRO A 323 −41.271 26.232 10.104 1.00 78.83 C ATOM 3384 N LEU A324 −42.426 28.454 12.899 1.00 80.72 N ATOM 3385 CA LEU A 324 −42.68929.050 14.233 1.00 81.22 C ATOM 3386 C LEU A 324 −41.580 30.013 14.6511.00 78.60 C ATOM 3387 O LEU A 324 −41.851 31.053 15.230 1.00 77.91 OATOM 3388 CB LEU A 324 −42.847 27.970 15.310 1.00 83.48 C ATOM 3389 CGLEU A 324 −44.017 27.003 15.125 1.00 90.04 C ATOM 3390 CD1 LEU A 324−43.821 25.727 15.948 1.00 91.90 C ATOM 3391 CD2 LEU A 324 −45.32927.707 15.474 1.00 93.47 C ATOM 3392 N ILE A 325 −40.334 29.656 14.3551.00 77.22 N ATOM 3393 CA ILE A 325 −39.186 30.504 14.689 1.00 76.61 CATOM 3394 C ILE A 325 −39.244 31.830 13.907 1.00 75.96 C ATOM 3395 O ILEA 325 −38.791 32.850 14.407 1.00 76.16 O ATOM 3396 CB ILE A 325 −37.83729.776 14.433 1.00 76.87 C ATOM 3397 CG1 ILE A 325 −37.701 28.520 15.3281.00 77.05 C ATOM 3398 CG2 ILE A 325 −36.654 30.701 14.689 1.00 73.66 CATOM 3399 CD1 ILE A 325 −36.611 27.536 14.870 1.00 70.46 C ATOM 3400 NTYR A 326 −39.803 31.815 12.690 1.00 76.28 N ATOM 3401 CA TYR A 326−39.934 33.054 11.884 1.00 76.44 C ATOM 3402 C TYR A 326 −40.978 34.01712.416 1.00 73.86 C ATOM 3403 O TYR A 326 −40.991 35.153 11.992 1.0077.86 O ATOM 3404 CB TYR A 326 −40.242 32.772 10.409 1.00 77.72 C ATOM3405 CG TYR A 326 −39.240 31.893 9.701 1.00 77.26 C ATOM 3406 CD1 TYR A326 −39.666 30.889 8.847 1.00 77.44 C ATOM 3407 CD2 TYR A 326 −37.86532.057 9.885 1.00 81.33 C ATOM 3408 CE1 TYR A 326 −38.764 30.077 8.1941.00 78.66 C ATOM 3409 CE2 TYR A 326 −36.961 31.248 9.236 1.00 83.58 CATOM 3410 CZ TYR A 326 −37.423 30.258 8.389 1.00 80.70 C ATOM 3411 OHTYR A 326 −36.541 29.451 7.742 1.00 81.84 O ATOM 3412 N CYS A 327−41.848 33.582 13.332 1.00 72.88 N ATOM 3413 CA CYS A 327 −42.782 34.49613.999 1.00 76.25 C ATOM 3414 C CYS A 327 −42.064 35.537 14.905 1.0078.27 C ATOM 3415 O CYS A 327 −42.684 36.508 15.330 1.00 79.91 O ATOM3416 CB CYS A 327 −43.827 33.729 14.805 1.00 77.89 C ATOM 3417 SG CYS A327 −44.970 32.742 13.778 1.00 82.26 S ATOM 3418 N ARG A 328 −40.77035.320 15.191 1.00 79.14 N ATOM 3419 CA ARG A 328 −39.919 36.324 15.8321.00 79.59 C ATOM 3420 C ARG A 328 −39.781 37.596 14.984 1.00 79.99 CATOM 3421 O ARG A 328 −39.581 38.680 15.524 1.00 79.93 O ATOM 3422 CBARG A 328 −38.515 35.771 16.078 1.00 77.25 C ATOM 3423 CG ARG A 328−38.423 34.646 17.130 1.00 76.09 C ATOM 3424 CD ARG A 328 −36.998 34.06717.220 1.00 77.31 C ATOM 3425 NE ARG A 328 −36.006 35.129 17.048 1.0075.58 N ATOM 3426 CZ ARG A 328 −35.537 35.945 17.985 1.00 81.23 C ATOM3427 NH1 ARG A 328 −35.953 35.859 19.252 1.00 74.46 N ATOM 3428 NH2 ARGA 328 −34.631 36.866 17.640 1.00 81.80 N ATOM 3429 N SER A 329 −39.88537.449 13.659 1.00 82.57 N ATOM 3430 CA SER A 329 −39.788 38.574 12.7431.00 84.20 C ATOM 3431 C SER A 329 −41.107 39.324 12.711 1.00 84.79 CATOM 3432 O SER A 329 −42.151 38.687 12.597 1.00 88.37 O ATOM 3433 CBSER A 329 −39.460 38.097 11.335 1.00 84.66 C ATOM 3434 OG SER A 329−39.465 39.190 10.429 1.00 90.44 O ATOM 3435 N PRO A 330 −41.076 40.67512.814 1.00 86.62 N ATOM 3436 CA PRO A 330 −42.339 41.415 12.685 1.0085.84 C ATOM 3437 C PRO A 330 −42.905 41.369 11.253 1.00 86.45 C ATOM3438 O PRO A 330 −44.103 41.466 11.079 1.00 85.67 O ATOM 3439 CB PRO A330 −41.960 42.851 13.080 1.00 85.22 C ATOM 3440 CG PRO A 330 −40.59742.764 13.669 1.00 85.83 C ATOM 3441 CD PRO A 330 −39.946 41.595 13.0451.00 85.56 C ATOM 3442 N ASP A 331 −42.031 41.216 10.256 1.00 87.75 NATOM 3443 CA ASP A 331 −42.440 41.127 8.854 1.00 88.35 C ATOM 3444 C ASPA 331 −43.160 39.831 8.516 1.00 88.54 C ATOM 3445 O ASP A 331 −44.18039.865 7.824 1.00 87.13 O ATOM 3446 CB ASP A 331 −41.225 41.268 7.9291.00 91.75 C ATOM 3447 CG ASP A 331 −40.585 42.640 8.000 1.00 99.65 CATOM 3448 OD1 ASP A 331 −41.152 43.526 8.660 1.00 101.57 O ATOM 3449 OD2ASP A 331 −39.507 42.825 7.387 1.00 108.72 O ATOM 3450 N PHE A 332−42.638 38.697 8.992 1.00 87.91 N ATOM 3451 CA PHE A 332 −43.306 37.3878.759 1.00 85.83 C ATOM 3452 C PHE A 332 −44.596 37.298 9.525 1.00 84.97C ATOM 3453 O PHE A 332 −45.583 36.811 9.006 1.00 85.85 O ATOM 3454 CBPHE A 332 −42.419 36.206 9.142 1.00 83.02 C ATOM 3455 CG PHE A 332−41.421 35.848 8.099 1.00 81.58 C ATOM 3456 CD1 PHE A 332 −40.184 36.4708.064 1.00 79.60 C ATOM 3457 CD2 PHE A 332 −41.719 34.884 7.141 1.0082.96 C ATOM 3458 CE1 PHE A 332 −39.251 36.138 7.092 1.00 83.90 C ATOM3459 CE2 PHE A 332 −40.794 34.541 6.159 1.00 81.77 C ATOM 3460 CZ PHE A332 −39.558 35.166 6.132 1.00 84.78 C ATOM 3461 N ARG A 333 −44.56737.772 10.764 1.00 85.64 N ATOM 3462 CA ARG A 333 −45.742 37.795 11.6181.00 86.23 C ATOM 3463 C ARG A 333 −46.875 38.603 10.964 1.00 85.68 CATOM 3464 O ARG A 333 −48.015 38.171 11.005 1.00 85.47 O ATOM 3465 CBARG A 333 −45.357 38.362 12.963 1.00 86.76 C ATOM 3466 CG ARG A 333−46.383 38.222 14.059 1.00 90.70 C ATOM 3467 CD ARG A 333 −45.671 38.26315.407 1.00 93.90 C ATOM 3468 NE ARG A 333 −44.710 39.377 15.472 1.00100.54 N ATOM 3469 CZ ARG A 333 −43.693 39.490 16.335 1.00 103.24 C ATOM3470 NH1 ARG A 333 −43.449 38.553 17.262 1.00 104.89 N ATOM 3471 NH2 ARGA 333 −42.899 40.566 16.271 1.00 102.64 N ATOM 3472 N ILE A 334 −46.53439.755 10.373 1.00 84.51 N ATOM 3473 CA ILE A 334 −47.472 40.580 9.5841.00 83.97 C ATOM 3474 C ILE A 334 −47.898 39.862 8.316 1.00 82.02 CATOM 3475 O ILE A 334 −49.077 39.871 7.963 1.00 86.53 O ATOM 3476 CB ILEA 334 −46.862 41.959 9.211 1.00 85.26 C ATOM 3477 CG1 ILE A 334 −46.77742.860 10.444 1.00 85.90 C ATOM 3478 CG2 ILE A 334 −47.692 42.668 8.1421.00 82.88 C ATOM 3479 CD1 ILE A 334 −45.791 44.015 10.287 1.00 88.19 CATOM 3480 N ALA A 335 −46.934 39.248 7.635 1.00 80.09 N ATOM 3481 CA ALAA 335 −47.202 38.470 6.433 1.00 79.44 C ATOM 3482 C ALA A 335 −48.19937.345 6.719 1.00 78.78 C ATOM 3483 O ALA A 335 −49.207 37.251 6.0571.00 78.15 O ATOM 3484 CB ALA A 335 −45.911 37.896 5.876 1.00 78.92 CATOM 3485 N PHE A 336 −47.896 36.511 7.714 1.00 78.39 N ATOM 3486 CA PHEA 336 −48.760 35.372 8.092 1.00 77.87 C ATOM 3487 C PHE A 336 −50.18435.751 8.427 1.00 77.70 C ATOM 3488 O PHE A 336 −51.116 35.205 7.8471.00 75.76 O ATOM 3489 CB PHE A 336 −48.198 34.602 9.301 1.00 77.26 CATOM 3490 CG PHE A 336 −46.852 33.943 9.072 1.00 77.36 C ATOM 3491 CD1PHE A 336 −46.408 33.562 7.796 1.00 78.61 C ATOM 3492 CD2 PHE A 336−46.024 33.695 10.152 1.00 76.74 C ATOM 3493 CE1 PHE A 336 −45.18032.962 7.621 1.00 76.21 C ATOM 3494 CE2 PHE A 336 −44.787 33.089 9.9741.00 78.36 C ATOM 3495 CZ PHE A 336 −44.372 32.724 8.700 1.00 76.78 CATOM 3496 N GLN A 337 −50.346 36.681 9.363 1.00 78.23 N ATOM 3497 CA GLNA 337 −51.691 37.124 9.792 1.00 79.51 C ATOM 3498 C GLN A 337 −52.54937.711 8.642 1.00 78.03 C ATOM 3499 O GLN A 337 −53.778 37.646 8.6941.00 78.24 O ATOM 3500 CB GLN A 337 −51.609 38.081 10.997 1.00 80.74 CATOM 3501 CG GLN A 337 −50.870 39.391 10.802 1.00 86.86 C ATOM 3502 CDGLN A 337 −50.613 40.113 12.143 1.00 89.20 C ATOM 3503 OE1 GLN A 337−51.317 39.877 13.127 1.00 97.34 O ATOM 3504 NE2 GLN A 337 −49.61040.989 12.177 1.00 94.24 N ATOM 3505 N GLU A 338 −51.900 38.271 7.6221.00 76.38 N ATOM 3506 CA GLU A 338 −52.591 38.694 6.411 1.00 74.47 CATOM 3507 C GLU A 338 −53.085 37.448 5.670 1.00 73.86 C ATOM 3508 O GLUA 338 −54.258 37.370 5.282 1.00 73.06 O ATOM 3509 CB GLU A 338 −51.65939.512 5.516 1.00 75.29 C ATOM 3510 CG GLU A 338 −52.343 40.279 4.3651.00 79.56 C ATOM 3511 CD GLU A 338 −52.695 39.442 3.113 1.00 85.28 CATOM 3512 OE1 GLU A 338 −52.500 38.207 3.087 1.00 91.20 O ATOM 3513 OE2GLU A 338 −53.176 40.048 2.131 1.00 89.12 O ATOM 3514 N LEU A 339−52.177 36.488 5.477 1.00 70.47 N ATOM 3515 CA LEU A 339 −52.475 35.2424.763 1.00 71.14 C ATOM 3516 C LEU A 339 −53.527 34.375 5.463 1.00 72.95C ATOM 3517 O LEU A 339 −54.255 33.650 4.793 1.00 74.46 O ATOM 3518 CBLEU A 339 −51.209 34.406 4.567 1.00 68.65 C ATOM 3519 CG LEU A 339−50.055 35.021 3.778 1.00 69.52 C ATOM 3520 CD1 LEU A 339 −48.805 34.1393.935 1.00 68.79 C ATOM 3521 CD2 LEU A 339 −50.411 35.225 2.315 1.0062.62 C ATOM 3522 N LEU A 340 −53.599 34.455 6.796 1.00 74.28 N ATOM3523 CA LEU A 340 −54.598 33.718 7.588 1.00 75.08 C ATOM 3524 C LEU A340 −55.848 34.573 7.901 1.00 76.80 C ATOM 3525 O LEU A 340 −56.57334.280 8.860 1.00 76.39 O ATOM 3526 CB LEU A 340 −53.960 33.174 8.8781.00 74.68 C ATOM 3527 CG LEU A 340 −52.763 32.233 8.701 1.00 73.84 CATOM 3528 CD1 LEU A 340 −52.150 31.887 10.048 1.00 73.83 C ATOM 3529 CD2LEU A 340 −53.167 30.976 7.982 1.00 73.24 C ATOM 3530 N CYS A 341−56.083 35.619 7.088 1.00 79.59 N ATOM 3531 CA CYS A 341 −57.280 36.4777.152 1.00 80.75 C ATOM 3532 C CYS A 341 −57.536 37.089 8.530 1.00 81.21C ATOM 3533 O CYS A 341 −58.613 36.911 9.114 1.00 81.97 O ATOM 3534 CBCYS A 341 −58.500 35.683 6.674 1.00 81.26 C ATOM 3535 SG CYS A 341−58.243 34.889 5.079 1.00 88.24 S ATOM 3536 N LEU A 342 −56.533 37.8099.031 1.00 81.84 N ATOM 3537 CA LEU A 342 −56.598 38.483 10.333 1.0082.60 C ATOM 3538 C LEU A 342 −56.370 39.993 10.158 1.00 83.35 C ATOM3539 O LEU A 342 −55.715 40.650 10.971 1.00 85.10 O ATOM 3540 CB LEU A342 −55.575 37.878 11.303 1.00 82.19 C ATOM 3541 CG LEU A 342 −55.51336.347 11.407 1.00 82.55 C ATOM 3542 CD1 LEU A 342 −54.415 35.913 12.3681.00 82.95 C ATOM 3543 CD2 LEU A 342 −56.851 35.782 11.838 1.00 83.45 CTER 3544 LEU A 342 HETATM 3545 C1 MAL A 401 −30.433 67.551 22.604 1.00116.54 C HETATM 3546 C2 MAL A 401 −29.508 68.271 21.613 1.00 116.67 CHETATM 3547 C3 MAL A 401 −28.286 67.466 21.250 1.00 115.67 C HETATM 3548C4 MAL A 401 −27.437 67.774 22.475 1.00 113.35 C HETATM 3549 C5 MAL A401 −28.222 67.326 23.742 1.00 114.16 C HETATM 3550 C6 MAL A 401 −27.46767.821 24.997 1.00 112.25 C HETATM 3551 O1 MAL A 401 −30.860 66.15222.481 1.00 118.82 O HETATM 3552 O2 MAL A 401 −30.196 68.896 20.430 1.00113.17 O HETATM 3553 O3 MAL A 401 −27.692 67.923 20.042 1.00 118.88 OHETATM 3554 O4 MAL A 401 −26.151 67.133 22.417 1.00 110.96 O HETATM 3555O5 MAL A 401 −29.640 67.723 23.796 1.00 116.03 O HETATM 3556 O6 MAL A401 −28.329 68.191 26.091 1.00 111.18 O HETATM 3557 C1′ MAL A 401−34.225 64.365 24.555 1.00 118.64 C HETATM 3558 C2′ MAL A 401 −34.06265.875 24.695 1.00 118.98 C HETATM 3559 C3′ MAL A 401 −33.053 66.49723.732 1.00 118.89 C HETATM 3560 C4′ MAL A 401 −31.773 65.653 23.5331.00 118.41 C HETATM 3561 C5′ MAL A 401 −32.192 64.169 23.329 1.00117.27 C HETATM 3562 C6′ MAL A 401 −31.044 63.164 23.154 1.00 114.49 CHETATM 3563 O1′ MAL A 401 −34.905 63.850 25.705 1.00 115.98 O HETATM3564 O2′ MAL A 401 −35.328 66.510 24.463 1.00 120.41 O HETATM 3565 O3′MAL A 401 −32.803 67.771 24.346 1.00 118.98 O HETATM 3566 O5′ MAL A 401−32.951 63.737 24.467 1.00 118.26 O HETATM 3567 O6′ MAL A 401 −31.31961.889 23.781 1.00 101.09 O HETATM 3568 S SO4 A 402 −30.549 40.587 1.8401.00 88.35 S HETATM 3569 O1 SO4 A 402 −31.012 40.657 0.449 1.00 91.49 OHETATM 3570 O2 SO4 A 402 −31.341 41.518 2.648 1.00 79.98 O HETATM 3571O3 SO4 A 402 −30.747 39.233 2.389 1.00 86.89 O HETATM 3572 O4 SO4 A 402−29.136 40.942 1.808 1.00 79.16 O HETATM 3573 S SO4 A 403 −34.529 39.15420.841 1.00 91.42 S HETATM 3574 O1 SO4 A 403 −35.518 40.193 21.131 1.0084.45 O HETATM 3575 O2 SO4 A 403 −33.296 39.334 21.612 1.00 93.22 OHETATM 3576 O3 SO4 A 403 −35.078 37.875 21.232 1.00 84.98 O HETATM 3577O4 SO4 A 403 −34.193 39.246 19.438 1.00 95.59 O HETATM 3578 S SO4 A 404−26.677 61.410 43.667 1.00 79.61 S HETATM 3579 O1 SO4 A 404 −26.22460.482 42.637 1.00 81.00 O HETATM 3580 O2 SO4 A 404 −27.968 62.00143.315 1.00 78.07 O HETATM 3581 O3 SO4 A 404 −26.852 60.655 44.907 1.0085.67 O HETATM 3582 O4 SO4 A 404 −25.700 62.487 43.819 1.00 76.33 OHETATM 3583 S SO4 A 405 −38.222 54.167 28.613 1.00 76.15 S HETATM 3584O1 SO4 A 405 −36.815 54.454 28.371 1.00 62.35 O HETATM 3585 O2 SO4 A 405−38.708 53.202 27.628 1.00 74.38 O HETATM 3586 O3 SO4 A 405 −38.39253.561 29.925 1.00 77.47 O HETATM 3587 O4 SO4 A 405 −38.983 55.41228.536 1.00 74.20 O HETATM 3588 S SO4 A 406 −14.626 46.742 34.955 1.00133.97 S HETATM 3589 O1 SO4 A 406 −14.905 46.888 33.527 1.00 132.94 OHETATM 3590 O2 SO4 A 406 −15.870 46.485 35.690 1.00 131.52 O HETATM 3591O3 SO4 A 406 −13.744 45.588 35.141 1.00 137.48 O HETATM 3592 O4 SO4 A406 −13.968 47.953 35.460 1.00 129.69 O HETATM 3593 S SO4 A 407 −39.37559.242 10.957 1.00 103.03 S HETATM 3594 O1 SO4 A 407 −38.112 58.59110.589 1.00 100.34 O HETATM 3595 O2 SO4 A 407 −40.365 59.029 9.899 1.00104.55 O HETATM 3596 O3 SO4 A 407 −39.865 58.663 12.211 1.00 107.47 OHETATM 3597 O4 SO4 A 407 −39.174 60.683 11.138 1.00 106.88 O HETATM 3598O17 CAU A 408 −33.477 10.957 8.170 1.00 50.96 O HETATM 3599 C16 CAU A408 −32.267 10.230 8.041 1.00 45.65 C HETATM 3600 C18 CAU A 408 −32.4788.951 7.225 1.00 51.24 C HETATM 3601 N19 CAU A 408 −33.702 8.250 7.6001.00 54.99 N HETATM 3602 C20 CAU A 408 −33.806 6.805 7.498 1.00 60.13 CHETATM 3603 C21 CAU A 408 −33.533 6.385 6.055 1.00 66.62 C HETATM 3604C22 CAU A 408 −35.184 6.350 7.988 1.00 59.87 C HETATM 3605 C15 CAU A 408−31.242 11.105 7.364 1.00 46.24 C HETATM 3606 O14 CAU A 408 −30.04910.367 7.182 1.00 51.01 O HETATM 3607 C13 CAU A 408 −28.931 10.857 6.5811.00 52.29 C HETATM 3608 C12 CAU A 408 −28.911 12.133 6.005 1.00 56.44 CHETATM 3609 C11 CAU A 408 −27.768 12.628 5.393 1.00 56.29 C HETATM 3610C10 CAU A 408 −26.608 11.875 5.332 1.00 54.90 C HETATM 3611 C8 CAU A 408−26.565 10.625 5.875 1.00 54.68 C HETATM 3612 C9 CAU A 408 −27.76810.108 6.517 1.00 53.44 C HETATM 3613 N7 CAU A 408 −25.593 9.657 5.9741.00 57.07 N HETATM 3614 C3 CAU A 408 −26.096 8.561 6.637 1.00 55.09 CHETATM 3615 C4 CAU A 408 −27.482 8.863 6.976 1.00 53.66 C HETATM 3616 C2CAU A 408 −25.589 7.338 7.009 1.00 54.54 C HETATM 3617 C1 CAU A 408−26.395 6.432 7.689 1.00 56.87 C HETATM 3618 C6 CAU A 408 −27.717 6.7318.006 1.00 56.28 C HETATM 3619 C5 CAU A 408 −28.269 7.948 7.652 1.0056.42 C HETATM 3620 C1 BU1 A 409 −26.142 41.424 19.127 1.00 52.74 CHETATM 3621 C2 BU1 A 409 −24.673 41.194 19.349 1.00 50.50 C HETATM 3622C3 BU1 A 409 −24.361 39.703 19.256 1.00 58.68 C HETATM 3623 C4 BU1 A 409−23.639 39.193 20.492 1.00 60.90 C HETATM 3624 O5 BU1 A 409 −26.44942.812 19.063 1.00 46.89 O HETATM 3625 O6 BU1 A 409 −23.397 37.79620.358 1.00 65.39 O HETATM 3626 C1 BU1 A 410 −25.492 26.523 17.727 1.0080.96 C HETATM 3627 C2 BU1 A 410 −25.259 26.818 16.240 1.00 75.39 CHETATM 3628 C3 BU1 A 410 −25.569 28.286 15.904 1.00 72.90 C HETATM 3629C4 BU1 A 410 −24.368 29.076 15.393 1.00 70.20 C HETATM 3630 O5 BU1 A 410−25.036 25.202 18.096 1.00 82.37 O HETATM 3631 O6 BU1 A 410 −23.72829.769 16.468 1.00 72.23 O HETATM 3632 C1 ACM A 411 −33.365 45.11623.775 1.00 72.09 C HETATM 3633 O ACM A 411 −34.580 45.100 23.784 1.0074.74 O HETATM 3634 N ACM A 411 −32.677 43.981 23.897 1.00 71.73 NHETATM 3635 C2 ACM A 411 −32.587 46.397 23.626 1.00 69.99 C HETATM 3636C1 CLR A 412 −36.292 27.653 −4.352 1.00 98.97 C HETATM 3637 C2 CLR A 412−36.571 29.133 −4.591 1.00 98.10 C HETATM 3638 C3 CLR A 412 −35.42529.793 −5.354 1.00 98.91 C HETATM 3639 C4 CLR A 412 −34.965 29.038−6.627 1.00 98.80 C HETATM 3640 C5 CLR A 412 −35.001 27.515 −6.513 1.0099.38 C HETATM 3641 C6 CLR A 412 −34.088 26.785 −7.209 1.00 99.72 CHETATM 3642 C7 CLR A 412 −34.017 25.259 −7.204 1.00 98.91 C HETATM 3643C8 CLR A 412 −35.275 24.631 −6.633 1.00 99.40 C HETATM 3644 C9 CLR A 412−35.651 25.377 −5.337 1.00 99.32 C HETATM 3645 C10 CLR A 412 −36.05026.846 −5.645 1.00 100.31 C HETATM 3646 C11 CLR A 412 −36.707 24.655−4.466 1.00 96.50 C HETATM 3647 C12 CLR A 412 −36.536 23.143 −4.365 1.0096.65 C HETATM 3648 C13 CLR A 412 −36.329 22.495 −5.719 1.00 100.10 CHETATM 3649 C14 CLR A 412 −35.100 23.138 −6.353 1.00 101.09 C HETATM3650 C15 CLR A 412 −34.782 22.237 −7.542 1.00 101.42 C HETATM 3651 C16CLR A 412 −35.217 20.837 −7.081 1.00 101.37 C HETATM 3652 C17 CLR A 412−35.995 21.000 −5.758 1.00 100.60 C HETATM 3653 C18 CLR A 412 −37.56222.726 −6.571 1.00 103.09 C HETATM 3654 C19 CLR A 412 −37.268 26.936−6.512 1.00 102.37 C HETATM 3655 C20 CLR A 412 −37.144 19.961 −5.6321.00 100.25 C HETATM 3656 C21 CLR A 412 −38.032 20.187 −4.406 1.00 98.50C HETATM 3657 C22 CLR A 412 −36.561 18.538 −5.657 1.00 101.01 C HETATM3658 C23 CLR A 412 −37.437 17.457 −5.012 1.00 103.22 C HETATM 3659 C24CLR A 412 −36.748 16.094 −5.099 1.00 105.36 C HETATM 3660 C25 CLR A 412−37.535 15.014 −5.853 1.00 107.48 C HETATM 3661 C26 CLR A 412 −36.57114.080 −6.591 1.00 107.73 C HETATM 3662 C27 CLR A 412 −38.402 14.192−4.899 1.00 106.61 C HETATM 3663 O1 CLR A 412 −35.828 31.158 −5.664 1.0099.70 O HETATM 3664 C1 CLR A 413 −52.083 29.442 3.198 1.00 84.76 CHETATM 3665 C2 CLR A 413 −52.244 30.951 3.100 1.00 85.75 C HETATM 3666C3 CLR A 413 −53.214 31.296 1.956 1.00 84.48 C HETATM 3667 C4 CLR A 413−54.606 30.718 2.233 1.00 82.59 C HETATM 3668 C5 CLR A 413 −54.42429.218 2.245 1.00 86.97 C HETATM 3669 C6 CLR A 413 −55.171 28.468 1.4001.00 86.01 C HETATM 3670 C7 CLR A 413 −55.077 26.968 1.309 1.00 88.00 CHETATM 3671 C8 CLR A 413 −54.340 26.362 2.487 1.00 89.33 C HETATM 3672C9 CLR A 413 −53.087 27.161 2.862 1.00 88.72 C HETATM 3673 C10 CLR A 413−53.399 28.636 3.214 1.00 85.54 C HETATM 3674 C11 CLR A 413 −52.26526.440 3.959 1.00 87.79 C HETATM 3675 C12 CLR A 413 −51.961 24.981 3.6161.00 90.38 C HETATM 3676 C13 CLR A 413 −53.219 24.202 3.283 1.00 90.23 CHETATM 3677 C14 CLR A 413 −53.904 24.933 2.142 1.00 92.32 C HETATM 3678C15 CLR A 413 −54.976 23.971 1.668 1.00 92.65 C HETATM 3679 C16 CLR A413 −54.282 22.616 1.827 1.00 93.79 C HETATM 3680 C17 CLR A 413 −53.01822.820 2.693 1.00 91.53 C HETATM 3681 C18 CLR A 413 −54.156 24.093 4.4991.00 87.86 C HETATM 3682 C19 CLR A 413 −54.039 28.774 4.595 1.00 82.64 CHETATM 3683 C20 CLR A 413 −52.772 21.624 3.653 1.00 91.51 C HETATM 3684C21 CLR A 413 −51.815 21.878 4.816 1.00 89.13 C HETATM 3685 C22 CLR A413 −52.233 20.430 2.857 1.00 91.91 C HETATM 3686 C23 CLR A 413 −52.84119.099 3.296 1.00 93.30 C HETATM 3687 C24 CLR A 413 −52.459 17.993 2.3111.00 92.99 C HETATM 3688 C25 CLR A 413 −51.938 16.746 3.002 1.00 92.39 CHETATM 3689 C26 CLR A 413 −52.059 15.541 2.076 1.00 94.36 C HETATM 3690C27 CLR A 413 −50.499 16.952 3.460 1.00 89.59 C HETATM 3691 O1 CLR A 413−53.334 32.684 1.607 1.00 81.53 O HETATM 3692 C1 CLR A 414 −41.70627.953 −4.419 1.00 114.97 C HETATM 3693 C2 CLR A 414 −41.768 29.448−4.709 1.00 114.73 C HETATM 3694 C3 CLR A 414 −40.664 29.838 −5.694 1.00114.23 C HETATM 3695 C4 CLR A 414 −40.711 29.022 −7.000 1.00 115.07 CHETATM 3696 C5 CLR A 414 −40.836 27.528 −6.714 1.00 115.29 C HETATM 3697C6 CLR A 414 −40.069 26.669 −7.418 1.00 115.44 C HETATM 3698 C7 CLR A414 −40.081 25.169 −7.226 1.00 116.17 C HETATM 3699 C8 CLR A 414 −41.30324.678 −6.473 1.00 115.82 C HETATM 3700 C9 CLR A 414 −41.567 25.570−5.252 1.00 114.14 C HETATM 3701 C10 CLR A 414 −41.815 27.052 −5.6521.00 114.08 C HETATM 3702 C11 CLR A 414 −42.685 24.996 −4.348 1.00113.90 C HETATM 3703 C12 CLR A 414 −42.459 23.526 −3.999 1.00 115.68 CHETATM 3704 C13 CLR A 414 −42.294 22.667 −5.238 1.00 115.89 C HETATM3705 C14 CLR A 414 −41.123 23.217 −6.042 1.00 116.05 C HETATM 3706 C15CLR A 414 −40.935 22.171 −7.143 1.00 117.57 C HETATM 3707 C16 CLR A 414−41.285 20.849 −6.450 1.00 117.86 C HETATM 3708 C17 CLR A 414 −41.87421.195 −5.060 1.00 116.61 C HETATM 3709 C18 CLR A 414 −43.602 22.724−6.051 1.00 114.74 C HETATM 3710 C19 CLR A 414 −43.197 27.247 −6.2851.00 114.74 C HETATM 3711 C20 CLR A 414 −42.937 20.193 −4.516 1.00116.12 C HETATM 3712 C21 CLR A 414 −43.284 20.508 −3.065 1.00 114.86 CHETATM 3713 C22 CLR A 414 −42.503 18.733 −4.603 1.00 115.06 C HETATM3714 C23 CLR A 414 −43.586 17.708 −4.216 1.00 115.00 C HETATM 3715 C24CLR A 414 −42.985 16.411 −3.662 1.00 113.44 C HETATM 3716 C25 CLR A 414−42.272 15.571 −4.726 1.00 113.32 C HETATM 3717 C26 CLR A 414 −40.86916.077 −5.018 1.00 111.52 C HETATM 3718 C27 CLR A 414 −42.204 14.107−4.309 1.00 112.05 C HETATM 3719 O1 CLR A 414 −40.674 31.263 −5.966 1.00114.05 O HETATM 3720 C1 PLM A 415 −57.959 33.303 4.949 1.00 94.70 CHETATM 3721 O2 PLM A 415 −57.047 33.732 4.211 1.00 99.49 O HETATM 3722C2 PLM A 415 −58.012 31.828 5.279 1.00 93.74 C HETATM 3723 C3 PLM A 415−59.131 31.125 4.492 1.00 92.47 C HETATM 3724 C4 PLM A 415 −58.64229.982 3.590 1.00 91.37 C HETATM 3725 C5 PLM A 415 −59.532 28.742 3.7011.00 89.98 C HETATM 3726 C6 PLM A 415 −59.310 27.816 2.505 1.00 88.93 CHETATM 3727 C7 PLM A 415 −60.003 26.467 2.677 1.00 86.74 C HETATM 3728C8 PLM A 415 −59.012 25.330 2.467 1.00 86.47 C HETATM 3729 C9 PLM A 415−59.709 23.971 2.504 1.00 88.96 C HETATM 3730 CA PLM A 415 −58.83722.893 3.156 1.00 90.43 C HETATM 3731 CB PLM A 415 −59.094 21.474 2.6221.00 94.25 C HETATM 3732 CC PLM A 415 −57.863 20.775 2.016 1.00 96.09 CHETATM 3733 CD PLM A 415 −57.471 19.483 2.752 1.00 100.00 C HETATM 3734CE PLM A 415 −56.635 18.544 1.872 1.00 101.35 C HETATM 3735 CF PLM A 415−56.391 17.207 2.576 1.00 101.14 C HETATM 3736 CG PLM A 415 −55.69516.199 1.664 1.00 99.15 C HETATM 3737 C35 12P A 416 −12.915 54.89717.807 1.00 109.60 C HETATM 3738 O34 12P A 416 −13.949 54.683 18.7771.00 110.80 O HETATM 3739 C33 12P A 416 −13.451 54.394 20.091 1.00108.13 C HETATM 3740 C32 12P A 416 −13.568 55.622 20.982 1.00 105.37 CHETATM 3741 O31 12P A 416 −12.299 56.022 21.539 1.00 101.39 O HETATM3742 C30 12P A 416 −12.283 57.340 22.133 1.00 102.00 C HETATM 3743 C2912P A 416 −13.231 57.476 23.346 1.00 99.93 C HETATM 3744 O28 12P A 416−14.580 57.575 22.874 1.00 94.22 O HETATM 3745 C27 12P A 416 −15.58058.132 23.720 1.00 91.37 C HETATM 3746 C26 12P A 416 −16.528 58.91222.820 1.00 89.49 C HETATM 3747 O25 12P A 416 −15.752 59.825 22.016 1.0091.31 O HETATM 3748 C24 12P A 416 −16.081 59.833 20.621 1.00 89.90 CHETATM 3749 C23 12P A 416 −14.998 60.574 19.838 1.00 88.02 C HETATM 3750O22 12P A 416 −14.838 61.904 20.352 1.00 86.45 O HETATM 3751 C21 12P A416 −15.839 62.838 19.944 1.00 77.75 C HETATM 3752 C20 12P A 416 −15.14663.916 19.149 1.00 82.56 C HETATM 3753 O19 12P A 416 −14.005 64.46219.834 1.00 79.81 O HETATM 3754 C18 12P A 416 −14.323 65.263 20.988 1.0086.00 C HETATM 3755 C17 12P A 416 −13.223 66.275 21.332 1.00 87.84 CHETATM 3756 O16 12P A 416 −11.998 65.967 20.653 1.00 93.31 O HETATM 3757C15 12P A 416 −11.011 65.354 21.493 1.00 96.79 C HETATM 3758 O HOH A 501−23.201 38.168 1.127 1.00 60.95 O HETATM 3759 O HOH A 502 −28.322 45.44519.106 1.00 62.27 O HETATM 3760 O HOH A 503 −25.663 51.540 11.557 1.0073.63 O HETATM 3761 O HOH A 504 −18.403 54.490 20.568 1.00 49.22 OHETATM 3762 O HOH A 505 −28.865 62.967 14.476 1.00 49.07 O HETATM 3763 OHOH A 506 −38.344 25.395 6.999 1.00 53.60 O HETATM 3764 O HOH A 507−24.235 47.669 28.279 1.00 58.12 O HETATM 3765 O HOH A 508 −29.56658.644 29.505 1.00 53.32 O HETATM 3766 O HOH A 509 −9.129 65.077 35.7881.00 53.82 O HETATM 3767 O HOH A 510 −31.588 56.235 5.270 1.00 54.83 OHETATM 3768 O HOH A 511 −33.767 −0.936 2.970 1.00 76.89 O HETATM 3769 OHOH A 512 −36.831 52.163 25.686 1.00 59.37 O HETATM 3770 O HOH A 513−20.293 51.784 5.369 1.00 57.17 O HETATM 3771 O HOH A 514 −33.710 48.03830.053 1.00 55.19 O HETATM 3772 O HOH A 515 −27.498 71.737 31.097 1.0072.77 O HETATM 3773 O HOH A 516 −38.419 75.003 17.409 1.00 63.18 OHETATM 3774 O HOH A 517 −13.008 52.291 33.720 1.00 54.29 O HETATM 3775 OHOH A 518 −25.417 49.449 2.526 1.00 84.88 O HETATM 3776 O HOH A 519−44.492 24.103 10.594 1.00 70.04 O HETATM 3777 O HOH A 520 −53.19234.656 −0.012 0.50 68.32 O HETATM 3778 O HOH A 521 −33.730 52.334 13.5311.00 71.71 O HETATM 3779 O HOH A 522 −19.401 44.894 11.284 1.00 82.98 OHETATM 3780 O HOH A 523 −20.706 −4.455 5.029 1.00 74.00 O HETATM 3781 OHOH A 524 −18.878 44.843 24.435 1.00 71.79 O HETATM 3782 O HOH A 525−26.266 47.546 12.074 1.00 96.88 O HETATM 3783 O HOH A 526 −35.81854.612 25.448 1.00 65.23 O HETATM 3784 O HOH A 527 −27.911 63.928 17.0981.00 56.73 O HETATM 3785 O HOH A 528 −34.136 24.300 12.374 1.00 64.85 OHETATM 3786 O HOH A 529 −31.485 13.403 16.427 1.00 55.45 O HETATM 3787 OHOH A 530 −33.229 46.525 27.509 1.00 62.72 O HETATM 3788 O HOH A 531−37.000 56.927 26.470 1.00 69.85 O HETATM 3789 O HOH A 532 −38.27223.691 9.991 1.00 74.37 O HETATM 3790 O HOH A 533 −23.738 45.700 30.2141.00 70.87 O HETATM 3791 O HOH A 534 −35.547 18.917 9.368 1.00 68.76 OHETATM 3792 O HOH A 535 −27.520 46.018 35.709 1.00 73.73 O HETATM 3793 OHOH A 536 −11.169 52.639 27.107 1.00 53.63 O HETATM 3794 O HOH A 537−35.161 36.603 6.920 1.00 84.70 O HETATM 3795 O HOH A 538 −13.331 64.63116.826 1.00 78.41 O HETATM 3796 O HOH A 539 −15.737 37.989 13.501 1.0074.20 O HETATM 3797 O HOH A 540 −17.612 49.885 19.741 1.00 67.69 OHETATM 3798 O HOH A 541 −28.871 56.041 4.930 1.00 62.51 O HETATM 3799 OHOH A 542 −18.100 62.470 17.192 1.00 64.11 O HETATM 3800 O HOH A 543−39.876 5.502 10.338 1.00 78.73 O HETATM 3801 O HOH A 544 −39.732 −5.317−4.531 1.00 75.67 O HETATM 3802 O HOH A 545 −19.865 59.621 17.222 1.0067.78 O HETATM 3803 O HOH A 546 −37.397 27.672 6.120 1.00 59.62 O HETATM3804 O HOH A 547 −19.199 49.833 44.410 1.00 55.49 O HETATM 3805 O HOH A548 −35.618 21.645 8.827 1.00 61.27 O CONECT 597 1283 CONECT 1229 1277CONECT 1277 1229 CONECT 1283 597 CONECT 2906 3635 CONECT 3535 3720CONECT 3545 3546 3551 3555 CONECT 3546 3545 3547 3552 CONECT 3547 35463548 3553 CONECT 3548 3547 3549 3554 CONECT 3549 3548 3550 3555 CONECT3550 3549 3556 CONECT 3551 3545 3560 CONECT 3552 3546 CONECT 3553 3547CONECT 3554 3548 CONECT 3555 3545 3549 CONECT 3556 3550 CONECT 3557 35583563 3566 CONECT 3558 3557 3559 3564 CONECT 3559 3558 3560 3565 CONECT3560 3551 3559 3561 CONECT 3561 3560 3562 3566 CONECT 3562 3561 3567CONECT 3563 3557 CONECT 3564 3558 CONECT 3565 3559 CONECT 3566 3557 3561CONECT 3567 3562 CONECT 3568 3569 3570 3571 3572 CONECT 3569 3568 CONECT3570 3568 CONECT 3571 3568 CONECT 3572 3568 CONECT 3573 3574 3575 35763577 CONECT 3574 3573 CONECT 3575 3573 CONECT 3576 3573 CONECT 3577 3573CONECT 3578 3579 3580 3581 3582 CONECT 3579 3578 CONECT 3580 3578 CONECT3581 3578 CONECT 3582 3578 CONECT 3583 3584 3585 3586 3587 CONECT 35843583 CONECT 3585 3583 CONECT 3586 3583 CONECT 3587 3583 CONECT 3588 35893590 3591 3592 CONECT 3589 3588 CONECT 3590 3588 CONECT 3591 3588 CONECT3592 3588 CONECT 3593 3594 3595 3596 3597 CONECT 3594 3593 CONECT 35953593 CONECT 3596 3593 CONECT 3597 3593 CONECT 3598 3599 CONECT 3599 35983600 3605 CONECT 3600 3599 3601 CONECT 3601 3600 3602 CONECT 3602 36013603 3604 CONECT 3603 3602 CONECT 3604 3602 CONECT 3605 3599 3606 CONECT3606 3605 3607 CONECT 3607 3606 3608 3612 CONECT 3608 3607 3609 CONECT3609 3608 3610 CONECT 3610 3609 3611 CONECT 3611 3610 3612 3613 CONECT3612 3607 3611 3615 CONECT 3613 3611 3614 CONECT 3614 3613 3615 3616CONECT 3615 3612 3614 3619 CONECT 3616 3614 3617 CONECT 3617 3616 3618CONECT 3618 3617 3619 CONECT 3619 3615 3618 CONECT 3620 3621 3624 CONECT3621 3620 3622 CONECT 3622 3621 3623 CONECT 3623 3622 3625 CONECT 36243620 CONECT 3625 3623 CONECT 3626 3627 3630 CONECT 3627 3626 3628 CONECT3628 3627 3629 CONECT 3629 3628 3631 CONECT 3630 3626 CONECT 3631 3629CONECT 3632 3633 3634 3635 CONECT 3633 3632 CONECT 3634 3632 CONECT 36352906 3632 CONECT 3636 3637 3645 CONECT 3637 3636 3638 CONECT 3638 36373639 3663 CONECT 3639 3638 3640 CONECT 3640 3639 3641 3645 CONECT 36413640 3642 CONECT 3642 3641 3643 CONECT 3643 3642 3644 3649 CONECT 36443643 3645 3646 CONECT 3645 3636 3640 3644 3654 CONECT 3646 3644 3647CONECT 3647 3646 3648 CONECT 3648 3647 3649 3652 3653 CONECT 3649 36433648 3650 CONECT 3650 3649 3651 CONECT 3651 3650 3652 CONECT 3652 36483651 3655 CONECT 3653 3648 CONECT 3654 3645 CONECT 3655 3652 3656 3657CONECT 3656 3655 CONECT 3657 3655 3658 CONECT 3658 3657 3659 CONECT 36593658 3660 CONECT 3660 3659 3661 3662 CONECT 3661 3660 CONECT 3662 3660CONECT 3663 3638 CONECT 3664 3665 3673 CONECT 3665 3664 3666 CONECT 36663665 3667 3691 CONECT 3667 3666 3668 CONECT 3668 3667 3669 3673 CONECT3669 3668 3670 CONECT 3670 3669 3671 CONECT 3671 3670 3672 3677 CONECT3672 3671 3673 3674 CONECT 3673 3664 3668 3672 3682 CONECT 3674 36723675 CONECT 3675 3674 3676 CONECT 3676 3675 3677 3680 3681 CONECT 36773671 3676 3678 CONECT 3678 3677 3679 CONECT 3679 3678 3680 CONECT 36803676 3679 3683 CONECT 3681 3676 CONECT 3682 3673 CONECT 3683 3680 36843685 CONECT 3684 3683 CONECT 3685 3683 3686 CONECT 3686 3685 3687 CONECT3687 3686 3688 CONECT 3688 3687 3689 3690 CONECT 3689 3688 CONECT 36903688 CONECT 3691 3666 CONECT 3692 3693 3701 CONECT 3693 3692 3694 CONECT3694 3693 3695 3719 CONECT 3695 3694 3696 CONECT 3696 3695 3697 3701CONECT 3697 3696 3698 CONECT 3698 3697 3699 CONECT 3699 3698 3700 3705CONECT 3700 3699 3701 3702 CONECT 3701 3692 3696 3700 3710 CONECT 37023700 3703 CONECT 3703 3702 3704 CONECT 3704 3703 3705 3708 3709 CONECT3705 3699 3704 3706 CONECT 3706 3705 3707 CONECT 3707 3706 3708 CONECT3708 3704 3707 3711 CONECT 3709 3704 CONECT 3710 3701 CONECT 3711 37083712 3713 CONECT 3712 3711 CONECT 3713 3711 3714 CONECT 3714 3713 3715CONECT 3715 3714 3716 CONECT 3716 3715 3717 3718 CONECT 3717 3716 CONECT3718 3716 CONECT 3719 3694 CONECT 3720 3535 3721 3722 CONECT 3721 3720CONECT 3722 3720 3723 CONECT 3723 3722 3724 CONECT 3724 3723 3725 CONECT3725 3724 3726 CONECT 3726 3725 3727 CONECT 3727 3726 3728 CONECT 37283727 3729 CONECT 3729 3728 3730 CONECT 3730 3729 3731 CONECT 3731 37303732 CONECT 3732 3731 3733 CONECT 3733 3732 3734 CONECT 3734 3733 3735CONECT 3735 3734 3736 CONECT 3736 3735 CONECT 3737 3738 CONECT 3738 37373739 CONECT 3739 3738 3740 CONECT 3740 3739 3741 CONECT 3741 3740 3742CONECT 3742 3741 3743 CONECT 3743 3742 3744 CONECT 3744 3743 3745 CONECT3745 3744 3746 CONECT 3746 3745 3747 CONECT 3747 3746 3748 CONECT 37483747 3749 CONECT 3749 3748 3750 CONECT 3750 3749 3751 CONECT 3751 37503752 CONECT 3752 3751 3753 CONECT 3753 3752 3754 CONECT 3754 3753 3755CONECT 3755 3754 3756 CONECT 3756 3755 3757 CONECT 3757 3756 MASTER 4550 16 19 4 0 18 6 3804 1 219 39 END

We claim:
 1. A composition comprising a polypeptide in crystalline form,wherein said polypeptide comprises: a G-protein coupled receptor (GPCR)comprising an IC3 loop containing a substitution that comprises theamino acid sequence of a lysozyme.
 2. A composition comprising a fusionprotein in crystalline form, wherein said fusion protein comprises, fromN-terminus to C-terminus: a) a first portion of a G-protein coupledreceptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4and TM5 regions of said GPCR; b) a domain comprising the amino acidsequence of a lysozyme; c) a second portion of said GPCR, wherein saidsecond portion comprises TM6 and TM7 regions of said GPCR.
 3. Thecomposition of claim 2, wherein said first and second portions of saidGPCR comprise the amino acid sequence of a naturally occurring GPCR. 4.The composition of claim 1, wherein said GPCR is a β2-adrenergicreceptor.
 5. The composition of claim 2, wherein said fusion protein isbound to a ligand for said GPCR.
 6. A composition comprising apolypeptide in crystalline form, wherein said polypeptide comprises,from N-terminus to C-terminus: a) a first portion of a G-protein coupledreceptor (GPCR), wherein said first portion comprises the amino acidsequence that is N-terminal to the IC3 loop of said GPCR; b) a domaincomprising the amino acid sequence of a lysozyme; c) a second portion ofsaid GPCR, wherein said second portion comprises the amino acid sequencethat is C-terminal to the IC3 loop of said GPCR.
 7. A crystalline fusionprotein which comprises a beta-2 adrenergic receptor and lysozyme. 8.The crystalline fusion protein of claim 7 wherein the lysozyme is a T4lysozyme sequence.
 9. The crystalline fusion protein of claim 7 whereinthe beta-2 adrenergic receptor has a E122W mutation.
 10. The crystallinefusion protein of claim 7 wherein the beta-2 adrenergic receptor has aE122W mutation and wherein the lysozyme is a T4 lysozyme sequence. 11.The crystalline fusion protein of claim 7 wherein the lysozyme is T4lysozyme sequence in place of the third intracellular loop of the beta-2adrenergic receptor.
 12. The crystalline fusion protein of claim 7wherein residues 231^(5.70) to 262^(6.24) are replaced by residues 2 to161 of T4 lysozyme, and the beta-2 adrenergic receptor is truncated atposition
 365. 13. The crystalline fusion protein of claim 7 furthercomprising a ligand non-covalently bound thereto.
 14. The crystallinefusion protein of claim 13 wherein the ligand is carazolol.